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Iris Pigment Epithelial Cells Express a Functional Lipopolysaccharide Receptor Complex
Abstract
Ocular pigment epithelial cells are hypothesized to play a role in the pathogenesis of acute anterior uveitis (AAU), where LPS activation of Toll-like receptors (TLRs) may serve as a trigger. In this study, the expression of LPS receptors in iris pigment epithelium (IPE) was determined.
RT-PCR, flow cytometry, Western blot, and immunohistochemistry were used to investigate the expression of the LPS receptor complex (TLR4, MD-2, and CD14) in primary human IPE. Cytokine secretion by LPS-treated IPE was measured by multiplex bead array and ELISA. The role of CD14 in modulating the LPS response was investigated by addition of soluble CD14 and by antibody neutralization studies. In vivo expression of CD14 was examined by immunohistochemistry and Western blot analysis.
IPE expressed TLR4, MD-2, and CD14 in vitro and secreted a panel of proinflammatory cytokines (IL-6, CXCL8, CXCL10, CCL2, CCL4, and CCL5) when stimulated with LPS. CXCL8 secretion by LPS-treated IPE was dependent on CD14 and TLR4. CD14 was detected in CD68+ cells in the iris by immunohistochemistry and in normal aqueous by Western blot analysis. Conclusions: IPE cells express a functional LPS receptor complex and are capable of promoting ocular inflammation through secretion of an array of proinflammatory mediators. CD14 was identified as a key molecule that modulated the LPS response in IPE.
... Whole cell lysates from IPE and RPE were prepared as previously described [23]. Briefly, cells were incubated for 30 minutes in ice cold lysis buffer (0.1% SDS, 0.5% NP-40 in 50 mM Tris-HCl pH 7.4) supplemented with a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail, Roche, Mannheim, Germany). ...
... The lack of TLR8 and 10 proteins in ocular pigment epithelial cells may reflect the low expression levels, which in turn may influence their response to PAMPs. IPE express TLR4 in vitro consistent with our earlier findings that these cells express a functional LPS receptor complex (TLR4, MD-2, and CD14) in vitro and secreted several pro-inflammatory cytokines including IL-6, IL-8, MCP-1, IP-10, MIP-1-beta and RANTES in response to LPS stimulation [23]. Here, we showed that RPE expressed all TLR transcripts except TLR7. ...
... Results are representative of three experiments. differential expression of CD14, a co-receptor of TLR4, between IPE and ARPE-19 [23]. However, expression of CD14 from donor-matched primary IPE and RPE was not significantly different between the two cell types (data not shown). ...
Background
Toll-like receptor (TLR) activation is hypothesized to contribute to inflammatory eye disease including uveitis, yet the distribution pattern of TLRs in human uveal tissues remains poorly described. The purpose of this study was to investigate the expression profile of TLRs in human iris pigment epithelial cells (IPE) at the gene and protein level and examine the effect of pathogen-associated molecular patterns (PAMPs), such as Pam3CSK4.3HCl, Poly(I:C), lipopolysaccharides (LPS from E. coli serotype O111:B4), Flagellin, MALP-2 (macrophage activating lipopeptide-2), Poly(U) and CpGODN2395 on the production of inflammatory mediators including interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) from human IPE and retinal pigment epithelial cells (RPE).
Methods
RT-PCR and Western blotting was employed to investigate the expression of TLRs 1–10 in primary IPE and RPE. Secretion of IL-8 or MCP-1 following treatment with PAMPs was measured by ELISA. The role of TLR2, TLR3 and TLR4 in mediating an inflammatory response was investigated using pharmacological TLR inhibitors.
Results
IPE and RPE expressed transcripts for TLR1-6 and 8–10; and proteins for TLR1-6 and 9. IPE secreted IL-8 or MCP-1 in response to Pam3CSK4.3HCl, Poly(I:C), LPS and MALP-2, whereas RPE produced IL-8 only after Poly(I:C), LPS or MALP-2 treatment. TLR inhibitors (OxPAPC, CI-095 and chloroquine) blocked IL-8 secretion in Poly(I:C), LPS or MALP-2-treated IPE and RPE.
Conclusions
Ocular pigment epithelial cells respond to PAMPs through activation of TLRs, particularly TLR2, TLR3 and TLR4. Expression of TLRs in human IPE cells provides a basis for responses to many ocular pathogens and their activation may be involved in the pathogenesis of ocular inflammation.
... 2019). ARPE-19 cells constitutively express toll-like receptor 4 (TLR4), which in conjunction with CD14 and MD-2 (myeloid differentiation protein-2), co-receptors also expressed in these cells, mediates LPS recognition (Chui et al., 2009;Mai et al., 2014). The formation of this complex leads to the production of proinflammatory mediators potentially involved in retinal degeneration and ocular inflammation (Elner et al., 2005;Chui et al., 2009). ...
... ARPE-19 cells constitutively express toll-like receptor 4 (TLR4), which in conjunction with CD14 and MD-2 (myeloid differentiation protein-2), co-receptors also expressed in these cells, mediates LPS recognition (Chui et al., 2009;Mai et al., 2014). The formation of this complex leads to the production of proinflammatory mediators potentially involved in retinal degeneration and ocular inflammation (Elner et al., 2005;Chui et al., 2009). In this work, this was observed by the increased levels of IL-6, IL-8, and nitrite in the supernatant of LPS-activated cells. ...
Uveitis is a group of sight-threatening ocular inflammatory disorders, whose mainstay of therapy is associated with severe adverse events, prompting the investigation of alternative treatments. The peptide melittin (MEL) is the major component of Apis mellifera bee venom and presents anti-inflammatory and antiangiogenic activities, with possible application in ophthalmology. This work aims to investigate the potential of intravitreal MEL in the treatment of ocular diseases involving inflammatory processes, especially uveitis. Safety of MEL was assessed in retinal cells, chick embryo chorioallantoic membranes, and rats. MEL at concentrations safe for intravitreal administration showed an antiangiogenic activity in the chorioallantoic membrane model comparable to bevacizumab, used as positive control. A protective anti-inflammatory effect in retinal cells stimulated with lipopolysaccharide (LPS) was also observed, without toxic effects. Finally, rats with bacille Calmette-Guerin- (BCG) induced uveitis treated with intravitreal MEL showed attenuated disease progression and improvement of clinical, morphological, and functional parameters, in addition to decreased levels of proinflammatory mediators in the posterior segment of the eye. These effects were comparable to the response observed with corticosteroid treatment. Therefore, MEL presents adequate safety profile for intraocular administration and has therapeutic potential as an anti-inflammatory and antiangiogenic agent for ocular diseases.
... The depletion of cytokines from the iris epithelial cells as revealed in the explants may represent an active release during the inflammatory process, which may gain support from the in vivo findings that only a small amount of IL-1β and IL-6 is localized in the iris epithelium after LPS treatment. Previous reports have shown a constitutive expression of TLR4 in human ciliary and iris epithelial cells (Brito et al., 2004;Chui et al., 2010). Moreover, LPS is able to stimulate production of an array of pro-inflammatory cytokines from primary human iris pigment epithelium (Chui et al., 2010), whereas primary rabbit pigmented ciliary epithelial cells have been shown to produce IL-6 upon stimulation of IL-1β (Fleisher et al., 2000), indicating the ability of these ocular cells in production of proinflammatory cytokines. ...
... Previous reports have shown a constitutive expression of TLR4 in human ciliary and iris epithelial cells (Brito et al., 2004;Chui et al., 2010). Moreover, LPS is able to stimulate production of an array of pro-inflammatory cytokines from primary human iris pigment epithelium (Chui et al., 2010), whereas primary rabbit pigmented ciliary epithelial cells have been shown to produce IL-6 upon stimulation of IL-1β (Fleisher et al., 2000), indicating the ability of these ocular cells in production of proinflammatory cytokines. These findings together indicate that ciliary and iris epithelial cells are contributing to immune surveillance in the eye, acting together with antigen presenting cells resident in these tissues as the first line defense against pathogenic substances. ...
The receptor for growth hormone-releasing hormone (GHRH-R) has been shown to upregulate specifically in the ciliary and iris epithelial cells and infiltrating cells in the aqueous humor in a rat model of acute anterior uveitis. Treatment with GHRHR-R antagonist alleviates significantly these inflammatory responses. Herein we investigated whether the ciliary and iris epithelial cells can respond directly to lipopolysaccharide (LPS) without the influences of circulating leukocytes to produce inflammatory mediators through a GHRH-R mediated mechanism. In explant cultures of rat ciliary body and iris, LPS caused a substantial increase of GHRH-R in 24 h. Immunohistochemistry showed a localization of TLR4, the receptor for LPS, and an elevated expression of IL-6 and IL-1β in ciliary and iris epithelial cells after LPS treatment. LPS also elevated the level of IL-1β, IL-6, and iNOS and increased secretion of IL-1β and IL-6 from the explants. The GHRH-R antagonist, MIA-602, suppressed the elevated expression of IL-1β and IL-6, and reduced the release of IL-6. Such effects were not seen for the GHRHR agonist, MR-409. When co-cultured with leukocytes, expression of GHRH-R in the ocular explants was further enhanced during LPS treatment. Our results demonstrate a direct action of LPS on ciliary and iris epithelial cells to produce pro-inflammatory factors through a GHRH-R mediated mechanism, and suggest a role of these epithelial cells, in addition to the resident antigen presenting cells, in immune surveillance of the eye. Infiltrating leukocytes may enhance these inflammatory responses by regulating GHRH-R in ciliary and iris epithelial cells, in addition to their functions of synthesizing proinflammatory cytokines.
... Bacteria are recognized by pattern recognition receptors known as Toll-like receptors (TLRs), which signal through the NF-kB pathway and upregulate the synthesis of cytokines and chemokines responsible for recruiting immune cells to the site of infection [9,10]. TLRs have been identified in many cells throughout the eye, including retinal pigment epithelial cells, astrocytes, corneal epithelium, iris epithelium, and Muller cells [11,12]. Studies have demonstrated the importance of TLR-mediated recognition of ocular pathogens during bacterial keratitis [13][14][15][16][17], but analysis of the role of TLRs in modulating posterior segment inflammation during bacterial infection is lacking. ...
... The fact that intraocular inflammation, although delayed, occurs at all in the absence of TLR2 suggests that pathogen recognition and the resulting response occurs by a redundant mechanism. As indicated earlier, other TLRs have been detected in cells throughout the eye [11,12] and have been shown to be important in ocular infection [13][14][15][16][17]. We also found diminished inflammation in B. cereus-infected eyes of TLR4 -/mice, results similar to that observed in infected TLR2 -/eyes (Novosad and Callegan, unpublished work). ...
Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2(-/-) mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to10(8) CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2(-/-) eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2(-/-) eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2(-/-) eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis.
... Iris pigment epithelial cells are key players in inflammation of the anterior segment of the eye, which defines anterior uveitis. They have a dual role, as key regulators of ocular immune privilege (20), and as the source of an array of inflammatory cytokines (52). Our data indicate that ZIKV is capable of productively infecting human iris pigment epithelial cells. ...
During recent Zika epidemics, adults infected with Zika virus (ZIKV) have developed organ-specific inflammatory complications. The most serious Zika-associated inflammatory eye disease is uveitis, which is commonly anterior in type, affecting both eyes and responding to corticosteroid eye drops. Mechanisms of Zika-associated anterior uveitis are unknown, but ZIKV has been identified in the aqueous humor of affected individuals. The iris pigment epithelium is a target cell population in viral anterior uveitis, and it acts to maintain immune privilege within the anterior eye. Interactions between ZIKV and human iris pigment epithelial cells were investigated with infectivity assays and RNA-sequencing. Primary cell isolates were prepared from eyes of 20 cadaveric donors, and infected for 24 hours with PRVABC59 strain ZIKV or incubated uninfected as control. Cytoimmunofluorescence, RT-qPCR on total cellular RNA, and focus-forming assays of culture supernatant showed cell isolates were permissive to infection, and supported replication and release of infectious ZIKV. To explore molecular responses of cell isolates to ZIKV infection at the whole transcriptome level, RNA was sequenced on the Illumina NextSeq 500 platform, and results were aligned to the human GRCh38 genome. Multidimensional scaling showed clear separation between transcriptomes of infected and uninfected cell isolates. Differential expression analysis indicated a vigorous molecular response of the cell to ZIKV: 7,935 genes were differentially expressed between ZIKV-infected and uninfected cells (FDR < 0.05), and 99% of 613 genes that changed at least two-fold were up-regulated. Reactome and KEGG pathway and Gene Ontology enrichment analyses indicated strong activation of viral recognition and defense, in addition to biosynthesis processes. A CHAT network included 6275 molecular nodes and 24 contextual hubs in the cell response to ZIKV infection. Receptor-interacting serine/threonine kinase 1 (RIPK1) was the most significantly connected contextual hub. Correlation of gene expression with read counts assigned to the ZIKV genome identified a negative correlation between interferon signaling and viral load across isolates. This work represents the first investigation of mechanisms of Zika-associated anterior uveitis using an in vitro human cell model. The results suggest the iris pigment epithelium mounts a molecular response that limits intraocular pathology in most individuals.
... LF aqueous flare value measured with laser flare meter in photon counts per millisecond (pc/ms) that TLR4 expression increases in the iris and in macrophages in the iris tissue during EIU[11]. Primary cultures of human iris pigment epithelium cells express a functional LPS receptor complex and can promote ocular inflammation through secretion of several proinflammatory cytokines[12]. ...
Background
Injections of lipopolysaccharide in animal models generate acute anterior uveitis (also known as endotoxin-induced uveitis), but the effects of lipopolysaccharide injection are unknown in humans. We describe an unusual case in which acute anterior uveitis was dramatically activated subsequent to botulinum toxin injection in a patient with Behçet’s disease but the acute anterior uveitis was satisfactorily attenuated by infliximab.
Case presentation
A 53-year-old Japanese man had normal ocular findings at his regularly scheduled appointment. He had been diagnosed as having incomplete-type Behçet’s disease 11 years before. Three years after the diagnosis he was given systemic infusions of 5 mg/kg infliximab every 8 weeks and he had not experienced a uveitis attack for 8 years with no treatment other than infliximab. Two days after the eye examination, he received intracutaneous botulinum toxin injections to treat axillary hyperhidrosis on both sides. Three hours after the injections, he noted rapidly increasing floaters in his right eye. Four days after the injections, his right eye showed severe acute anterior uveitis with deteriorated aqueous flare and anterior vitreous opacity. He received his scheduled infliximab injection, and the right acute anterior uveitis immediately attenuated.
Conclusions
Botulinum toxin may have clinical effects similar to those of lipopolysaccharide in endotoxin-induced uveitis models. To the best of our knowledge, this is the first report to suggest that botulinum toxin may trigger acute anterior uveitis, although the precise mechanism is still unclear.
... Until now, it was assumed that molecular mimicry, pro-inflammatory cytokines, and Toll-like receptors (TLRs) trigger host innate immune response to microbial components [2,5]. Thus, bacterial lipopolysaccharide, by TLR-4, stimulates the iris pigment epithelial cells and activates the secretion of proinflammatory chemokines (including IL-8) [6]. When antigen-presenting cells from perivascular areas of the uvea are targeted by TLR-4, they are recruited into the uvea. ...
Hypothesis:Abnormal Vitamin D (Vit D) level could have consequences on the immuno-inflammatory processes in Ankylosing Spondylitis (AS).
Aim:The purpose of this study was to analyze the role of Vitamin D in the interplay between immune and inflammation effectors in AS associated-Acute Anterior Uveitis (AAU).
Methods and Results:25-hydroxyvitamin D (Vit D), LL-37 peptide, IL-8 and Serum Amyloid A (SAA) were identified and quantified in the serum/ plasma of thirty-four AS patients [eleven AS patients presenting AAU (AAU AS patients) and twenty-three AS patients without AAU (wAAU AS patients)] and eighteen healthy individuals (Control) using enzyme-linked immunosorbent assay. Acute-phase SAA level was significantly higher in AS patients compared to Controls. Contrary with wAAU AS patients, significantly elevated levels of IL-8, and diminished levels of Vit D characterized AAU AS patients. Regarding LL-37, its level decreased concomitantly with the level of Vit D. When AS patients were subgrouped based on AAU presence or on Vit D level, important associations between immuno-inflammatory assessed markers and AS features were noticed. Generally, Vit D levels were associated indirectly with leukocytes/ neutrophils number or with ESR, CRP, and Fibrinogen levels. The levels of SAA and IL-8 associated directly with AAU or with AAU relapses, especially in AS patients with Vit D insufficiency, while SAA associated directly with infection/ inflammatory markers and with disease activity indexes or with the degree of functional limitation.
Discussion:Altered levels of Vit D affect the balance between LL-37, IL-8 and SAA, suggesting an association with AAU, an extra-articular manifestation of AS.
Abbreviations:Vit D = Vitamin D, AS = Ankylosing Spondylitis, AAU = Acute Anterior Uveitis, AAU AS = AS patients with AAU, wAAU AS = AS patients without AAU, SSZ = Sulphasalazine, Leu = Leukocytes, Neu = Neutrophils.
... 4,5 For instance, studies have shown that singlenucleotide polymorphisms (SNPs) in TNF and their associated receptors are associated with AAU. 6 The levels of CCL2, CCL5, and their receptors have also been found to be significantly correlated with the severity of inflammation in AAU patients. 7 Moreover, the influence of genetic polymorphisms on uveitis might depend on the patient's HLA-B27 status. 8 Thus, there is robust evidence showing that genetic factors have an important role in the development of AAU. ...
PurposeCD59 complement regulator and complement factor H (CFH) have important roles in complement activation pathways, which are known to affect the development of uveitis. The present study was performed to investigate whether an association exists between CD59 and CFH genetic polymorphisms and acute anterior uveitis (AAU).MethodsA total of 600 individuals (300 patients diagnosed with AAU and 300 healthy controls) were recruited for this case-control study. Five single-nucleotide polymorphisms (SNPs) in CD59 (rs831626, rs12272807, rs831625, rs11585, and rs12576440) and CFH-rs1065489 were genotyped using Sequenom MassARRAY technology. Allele and genotype frequencies were statistically compared between patients and controls using χ(2) test. Analyses were stratified for gender, human leukocyte antigen (HLA)-B27, and ankylosing spondylitis (AS) status.ResultsNo significant association was found between any of the six polymorphisms and AAU. In HLA-B27-negative AAU patients, the frequencies of the G allele and GG homozygosity were lower in CD59-rs831626 when compared with controls (P=0.032). There were also significant decreases in the frequencies of T allele and TT homozygosity in CFH-rs1065489 in AAU patients with AS compared with controls (P=0.002). Furthermore, the frequencies of the T allele and TT homozygosity in CFH-rs1065489 were lower in the AAU male patients with AS compared with controls (P=0.015).Conclusion
Our results revealed that SNPs CD59-rs831626 and CFH-rs1065489 were associated with the susceptibility of AAU. The influence on AAU could be gender specific and dependent on the HLA-B27 and AS status. No positive results were found in the overall group.Eye advance online publication, 15 July 2016; doi:10.1038/eye.2016.146.
... For example, studies have shown that single-nucleotide polymorphisms (SNPs) in TNF and TNFRs are significantly correlated with AAU diagnosis [5]. The levels of CC chemokine ligand (CCL) 2, CCL5, and their receptors have been associated with AAU inflammation severity [6]. Moreover, the influence of genetic polymorphisms on uveitis might depend on HLA-B27 status [7]. ...
Background:
Complement factor H (CFH) related proteins (CFHRs) play important roles in complement activation pathways, while previous studies have only shown that CFH can affect the development of uveitis. In this study, we investigated the potential associations between one of single-nucleotide polymorphisms (SNPs) in the CFHR2 gene with acute anterior uveitis (AAU).
Methods:
A total of 571 subjects, 283 patients diagnosed with AAU and 288 healthy adult controls, were recruited for this case-control study. CFHR2-rs2986127 was detected using the Sequenom MassARRAY technology.
Results:
The stratified analyses for AAU patients with AS revealed a reduced frequency of the A allele in CFHR2-rs2986127 when compared to controls (P = 0.033, OR = 0.563, 95%CI = 330 to 0.960). Further stratified analyses revealed a similar significant reduced frequency in male AAU patients with AS when compared with male controls (P = 0.036, OR = 0.514, 95%CI = 0.274 to 0.965), and in AAU patients without posterior segment involvement when compared with controls (P = 0.048) as well.
Conclusions:
This study reveals an association between CFHR2-rs2986127 and AAU diagnosis, especially in gender, AS status and other clinical signs, such as posterior segment involvement. Our results may further enrich the growing understanding of uveitis genetics, and raise clinical diagnostic accuracy of this disease.
... We purchased human primary IPE and RPE cells with epithelial cell medium from ScienCell Research Laboratories (San Diego, CA) and cultured them as previously described. 35 Two control cell lines, THP1 (a human acute monocytic leukaemia cell line) and SKBR3 (a breast cancer cell line) were donated in the form of freshly extracted RNA by the ocular immunology group at the University of NSW, Kensington. ...
Purpose:
The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye.
Methods:
We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF).
Results:
Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines.
Conclusion:
This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions.
... TLRs, at the ocular surface, such as the corneal and conjunctival epithelia, might contribute to innate-immune responses that protect the eye from microbial infection [27]. Although intraocular cells, such as uveal epithelial cells [28], retinal pigmental epithelium cells [29,30], retinal photoreceptor cells [31], and astrocytes [32][33][34] express TLRs, their responses to endogenous substances, such as DAMPs, in the eye and their roles in the pathogenesis of noninfectious ocular diseases remain largely unknown. ...
It is largely unknown how invading autoreactive T cells initiate the pathogenic process inside the diseased organ in organ-specific autoimmune disease. In this study, we used a chronic uveitis disease model in mice-EAU-induced by adoptive transfer of uveitogenic IRBP-specific T cells and showed that HMGB1, an important endogenous molecule that serves as a danger signal, was released rapidly from retinal cells into the ECM and intraocular fluid in response to IRBP-specific T cell transfer. HMGB1 release required direct cell-cell contact between retinal cells and IRBP-specific T cells and was an active secretion from intact retinal cells. Administration of HMGB1 antagonists inhibited severity of EAU significantly via mechanisms that include inhibition of IRBP-specific T cell proliferation and their IFN-γ and IL-17 production. The inflammatory effects of HMGB1 may signal the TLR/MyD88 pathway, as MyD88(-/-) mice had a high level of HMGB1 in the eye but did not develop EAU after IRBP-specific T cell transfer. Our study demonstrates that HMGB1 is an early and critical mediator of ocular inflammation initiated by autoreactive T cell invasion.
Uveitis is a common manifestation of post-Ebola syndrome, associated with persistence of Ebola virus (EBOV; Zaire ebolavirus) inside the eye. The iris and retinal pigment epithelia are key components of the blood-ocular barriers, but have the capacity to act as hosts for microorganisms. We investigated the ability of EBOV to productively infect these cell populations. Donor-matched human iris and retinal pigment epithelial isolates (n = 5) were infected with EBOV at a multiplicity of infection of 1 for up to 72 hours. Parallel cultures were infected with Reston virus (RESTV; Reston ebolavirus) or Zika virus (ZIKV), or held uninfected under the same conditions. Viral transcript expression by RT-qPCR on total cellular RNA, cytoimmunofluorescence, and assays of 50% tissue culture infected dose of culture supernatant showed that both iris and retinal pigment epithelial isolates were permissive to infection, and supported replication and release of EBOV, as well as RESTV and ZIKV. However, in comparison to cells isolated from iris, those from retina demonstrated obvious EBOV-induced cytopathic effect, had higher intracellular EBOV nucleoprotein transcript, expressed intracellular EBOV protein more widely, and released EBOV at higher titer. Comparable results were obtained for isolates infected with RESTV and ZIKV. Consistent with observations of retinal pigment epithelial scars in Ebola survivors, our results suggest that an early event in post-Ebola uveitis is infection of the retinal pigment epithelium. Relative susceptibility of retinal pigment epithelial cells to infection with RESTV and ZIKV, as well as EBOV, implies this phenomenon may relate to a cell-specific attribute, such as high phagocytic activity.
Purpose:
To describe the clinical profile of patients presenting with uveitis following COVID-19 infection at a tertiary care eye hospital in South India.
Methods:
In this retrospective chart review, all consecutive cases presenting with an acute episode of intraocular inflammation and a history of COVID-19 infection diagnosed within the preceding 6 weeks, between March 2020 and September 2021, were included. Data retrieved and analyzed included age, sex, laterality of uveitis, and site of inflammation. The diagnosis was categorized based on the SUN working group classification criteria for uveitis. Details regarding clinical features, investigations, ophthalmic treatment given, response to treatment, ocular complications, and status at last visit were also accessed. Statistical analysis of demographical data was done using Microsoft Excel 2019.
Results:
Twenty-one eyes of 13 patients were included in this hospital-based retrospective observational study. The study included six male and seven female patients. The mean age was 38 ± 16.8 years. Eight patients had bilateral involvement. Seven patients were diagnosed with anterior uveitis, three with intermediate uveitis, one with posterior uveitis, and two with panuveitis. All patients responded well to treatment and were doing well at their last visit. Two patients had complications that necessitated surgical treatment, following which they recovered good visual outcomes.
Conclusion:
With prompt diagnosis and appropriate management, all the patients with uveitis post-COVID-19 infection recovered with good visual outcomes. Thus, ophthalmologists must be aware of the possible uveitic manifestations following even uneventful COVID-19 infection.
The uveal tract consists of the iris, the ciliary body and the choroid; these three distinct tissues form a continuous layer within the eye. Uveitis refers to inflammation of any region of the uveal tract. Despite being grouped together anatomically, the iris, ciliary body and choroid are distinct functionally, and inflammatory diseases may affect only one part and not the others. Cellular structure of tissues direct their function, and understanding the cellular basis of the immune environment of a tissue in health, the “steady state” on which the perturbations of disease are superimposed, is vital to understanding the pathogenesis of those diseases. A contemporary understanding of the immune system accepts that haematopoietic and yolk sac derived leukocytes, though vital, are not the only players of importance. An array of stromal cells, connective tissue cells such as fibroblasts and endothelial cells, may also have a role in the inflammatory reaction seen in several immune-mediated diseases. In this review we summarise what is known about the cellular composition of the uveal tract and the roles these disparate cell types have to play in immune homeostasis. We also discuss some unanswered questions surrounding the constituents of the resident leukocyte population of the different uveal tissues, and we look ahead to the new understanding that modern investigative techniques such as single cell transcriptomics, multi-omic data integration and highly-multiplexed imaging techniques may bring to the study of the uvea and uveitis, as they already have to other immune mediated inflammatory diseases.
Purpose
Lipopolysaccharide (LPS) can activate Toll-like receptor 4 (TLR4) and increase the expression of CXCL1 and CXCL2, the potent neutrophils chemoattractants, in various cell types. These effects have not been previously reported in the uveal melanocytes. This study was designed to investigate the effects of LPS on the activation of TLR4 and expression of CXCL1/CXCL2 in cultured human uveal melanocytes and the relevant signal pathways.
Methods
: Effects of LPS on the expression of TLR4 were tested using real time PCR, flow cytometry and fluorescence immunostaining. Effects of LPS-induced expression/secretion of CXCL1/CXCL2 were studied using real time PCR in cell lysates and ELISA in conditioned media of cultured uveal melanocytes. Activated NF-κB and phosphorylated MAPK signals were tested in cells with and without LPS treatment using flow cytometry. Effects of various signal inhibitors on p38, ERK1/2, JNK1/2 and NF-κB on the secretion of CXCL1/CXCL2 were tested by ELISA. The effects of neutralized antibodies of CXCL1/CXCL2 on the severity of LPS-induced uveitis were tested in a mouse model.
Results
LPS stimulation increased the expression of TLR4 mRNA and protein in culture uveal melanocytes. Constitutive secretion of CXCL1/CXCL2 were detected in uveal melanocytes and were significantly increased dose- and time-dependently by LPS stimulation. LPS mainly increased the activated NF-κB and phosphorylated JNK1/2. LPS-induced expression of CXCL1/CXCL2 were blocked by NF-κB and JNK1/2 inhibitors. The severity of LPS-induced uveitis was significantly inhibited by neutralizing antibody to CXCL1/CXCL2
Conclusions
: This is the first report on the LPS-induced expression of CXCL1 and CXCL2 by uveal melanocytes via the activation of TLR4. These results suggest that uveal melanocytes may play a role in the immune reaction that eliminates the invading pathogens. Conversely, an excessive LPS-induced inflammatory reaction may also lead to the development of inflammatory ocular disorders, such as non-infectious uveitis.
Purpose: To determine the anti-inflammatory effects of dehydroxymethylepoxyquinomicin (DHMEQ), a nuclear factor-κB (NF-κB) inhibitor, on endotoxin-induced uveitis (EIU) in rats.
Methods: EIU was induced by a subcutaneous injection of lipopolysaccharide (LPS) in Lewis rats. DHMEQ was injected intraperitoneally concurrently with the LPS. Aqueous humor was collected 24 h after the LPS injection. Isolated peritoneal exudate cells (PECs) were exposed to LPS with or without DHMEQ to determine the production of TNF-α, IL-6, and MCP-1.
Results: DHMEQ significantly reduced the number of infiltrating cells, and the concentrations of proteins, TNF-α, and IL-6 in the aqueous humor. DHMEQ suppressed the production of TNF-α, IL-6, and MCP-1 from PECs. Immunochemistry revealed a reduction in the translocation of the NF-κB p65 into the nuclei in DHMEQ-exposed PECs.
Conclusions: The results indicate that DHMEQ has anti-inflammatory effects on EIU and may be a promising agent to treat intraocular inflammation.
Today we are able to differentiate approximately a hundred entities of intraocular inflammations and group them into anatomical types like anterior, intermediate, posterior, and panuveitis. This book will cover most of the reported entities. A few of these entities have highly typical clinical findings, so that the specialist easily may be able to determine the entity, leading to therapy. But a lot of entities develop similarities in their clinical appearance (similar endothelial precipitates, similar chorioretinal infiltrations, similar retinal necrosis) and also similar complications (elevated intraocular pressure, cataract formation, and macular edema).
A bstract
Uveitis involves acute, recurrent or chronic inflammation of the uvea, and occurs when the normal state of ocular immune privilege has broken down. Accumulating evidence implicates the role of microbial triggers in the development of various forms of immune‐mediated uveitis in addition to its causative role in infectious uveitis. Toll‐like receptors (TLRs) are the most important pattern‐recognition receptors of the innate immune system that recognize pathogen‐associated molecular patterns of microbes. Activation of TLRs by pathogen‐associated molecular patterns leads to the induction of an inflammatory cascade and activation of both innate and adaptive arms of the immune response. TLRs have been implicated in the pathogenesis of various inflammatory diseases, including uveitis. This review provides an update on recent progress in TLR research and uveitis, specifically summarizing new evidence for the role of TLRs in anterior uveitis. There have been important observations from studies involving human ocular tissue, clinical uveitis and from experimental animal models of uveitis, such as endotoxin‐induced uveitis. The ‘Toll rush’ has certainly gained momentum, and future advances in this field have the potential for selectively targeting the TLR pathway and ultimately translating into better therapies for patients with sight‐threatening uveitis.
Acute anterior uveitis (AAU) is the most common form of uveitis; however, while it is presumed to have an immunological basis, the precise underlying etiology remains elusive. Toll-like receptors (TLRs) have a key role in linking innate and adaptive immunity, thereby forming a molecular bridge between microbial triggers and the development of AAU. The purpose of this study was to investigate the role of TLR2 and TLR4 gene polymorphisms in the pathogenesis of AAU.
The study comprised 225 confirmed cases of idiopathic or human leukocyte antigen (HLA) B27 (subtypes B*2701-2759; HLA-B27)-related AAU and 2,534 population-based controls from the Blue Mountains Eye Study. All participants were of Anglo-Celtic descent. Blood samples were collected for DNA extraction and genotyping. A total of 16 single nucleotide polymorphisms (SNPs) were selected for analysis and either directly genotyped or imputed to cover the common variations within the TLR genes. Data were analyzed at the allelic, genotypic and haplotypic levels.
Control subjects were significantly older than case subjects (p<0.0001). There was no significant difference in the gender composition between the case and control cohorts (p=0.18). One TLR2 SNP (rs11938228) was found to be associated with AAU at the allelic level (OR=1.28; p=0.017); however this association did not remain following adjustment for age and sex (p=0.067). None of the SNPs at the TLR4 locus were found to differ significantly between cases or controls, irrespective of adjustment for age and gender.
This study has confirmed that common TLR variants of moderate effect size do not predispose to AAU, undermining the implication of reported mutations in the selective perturbations of TLR expression and function evident in AAU.
The aqueous humor (AH) contains numerous immunosuppressive molecules that contribute to the ocular immune privilege. Here, we mimic an inflammatory environment to analyze the inhibitory effects of the AH on lipopolysaccharide (LPS)-induced maturation of dendritic cells (DC).
Different concentrations of AH were added to dendritic cell cultures together with LPS. Dendritic cell surface markers CD80, CD86, and MHC-II were assessed by use of flow cytometry. Endocytic capability and mixed lymphocyte reaction were measured as functional maturation.
AH inhibited LPS-induced DC maturation, resulting in down-regulated expression of CD80, CD86, MHC-II, enhancement of endocytic capacity, and reduced T cell activation. Neutralizing transforming growth factor beta 2 (TGF-β(2)) in AH can totally reverse the inhibitory effect. Treatment with prostaglandin E2 (PGE(2)) antagonist alone had no effect on DC maturation. However, blocking of both TGF-β(2) and PGE(2) in the AH resulted in synergistic suppression of the inhibiting effect of AH.
These results reveal that TGF-β(2) in the AH is of crucial importance in maintaining DC in the immature state. Further experiments will clarify the immune role of PGE(2) in AH.
Endotoxin shock is the result of activation of the immune system by endotoxin/LPS, a component of Gram-negative bacteria. CD14, a GPI-anchored glycoprotein expressed strongly by monocyte/macrophages, is one of several receptors for endotoxin/LPS. The role of CD14 in bacterial-induced and LPS-induced shock was tested in CD14-deficient mice produced by gene targeting in embryonic stem cells. CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria or LPS; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected. Surprisingly, CD14-deficient mice also showed dramatically reduced levels of bacteremia, suggesting an unexpected role for CD14 in the dissemination of Gram-negative bacteria.
To investigate the expression and polymorphisms of Toll-like receptor (TLR)-2 and -4 in the peripheral neutrophils and monocytes of patients with acute anterior uveitis (AAU).
Nine patients with active AAU and nine age- and sex-matched healthy control subjects were studied. TLR2 and -4 protein expression on CD16(+) neutrophils and CD14(+) monocytes were studied by flow cytometry. TLR function was investigated by whole-blood stimulation with lipopolysaccharide and peptidoglycan for TLR4 and -2 activation, respectively. Proinflammatory cytokine production in response to TLR stimulation was determined by multiplex cytokine bead arrays of the culture supernatant. TLR2 and -4 genotypes were determined by RFLP-PCR.
A significant reduction in the levels of TLR2 expression was observed on the neutrophils and monocytes of patients with active AAU compared with that of healthy control subjects (P < 0.05). IL-6 and IFN-gamma production stimulated by TLR4 activation was significantly reduced in patients with AAU, compared with that in healthy control subjects (P < 0.05). In contrast, significantly increased production of IL-1beta in response to TLR2 stimulation was observed in patients with AAU (P < 0.05). There was no correlation between the TLR2 or -4 genotypes and the observed differential functional responses to TLR stimulation.
There is a selective perturbation in the expression and function of TLR2 and -4, which could be consistent with a state of endotoxin tolerance, in patients with active AAU. The results support a role for microbial triggers and TLRs in the pathogenesis of AAU.
In a one-stage, interleukin-2 (IL-2), limiting-dilution analysis, peripheral blood mononuclear cells from patients with uveitis and normal control subjects were assayed for S-antigen specific, tetanus-specific, and in vivo activated helper T cells. Controls subjects consistently demonstrated tetanus-specific responses, but neither in vivo activation nor S-antigen specific helper T cell responses were seen. Patients with active forms of diffuse, posterior, and anterior uveitis were found to have significant frequencies of both in vivo activated and S-antigen specific helper T cells in their peripheral blood. These data show that patients with certain forms of uveitis have a measurable frequency of lymphocytes in the peripheral immunologic compartment capable of secreting IL-2 in response to autologous presentation of ocular autoantigen (S-antigen). Limiting-dilution analysis techniques, generating minimal responder cell frequency estimates and distinct IL secretion patterns, may provide an index of disease activity and critical information about the mechanism(s) of ocular inflammation.
Studies were undertaken to examine hepatocyte CD14 expression during endotoxemia. Our results show that lipopolysaccharide (LPS) treatment in vivo caused a marked upregulation in CD14 mRNA and protein levels in rat hepatocytes. Detectable increases in mRNA were seen as early as 1.5 h after LPS treatment; these increases peaked at 20-fold by 3 h and returned to baseline levels by 24 h. In situ hybridization localized the CD14 mRNA expression to hepatocytes both in vitro and in vivo. Increases in hepatic CD14 protein levels were detectable by 3 h and peaked at 12 h. Hepatocytes from LPS-treated animals expressed greater amounts of cell-associated CD14 protein, and more of the soluble CD14 was released by hepatocytes from LPS-treated rats in vitro. The increases in hepatocyte CD14 expression during endotoxemia occurred in parallel to increases of CD14 levels in plasma. To provide molecular identification of the hepatocyte CD14, we cloned the rat liver CD14 cDNA. The longest clone consists of a 1,591-bp insert containing a 1,116-bp open reading frame. The deduced amino acid sequence is 372 amino acids long, has 81.8 and 62.8% homology to the amino acid sequences of mouse and human CD14, respectively, and is identical to the rat macrophage CD14. The expressed CD14 protein from this clone was functional, as indicated by NF-kappaB activation in response to LPS and fluorescein isothiocyanate-LPS binding in CHO cells stably transfected with rat CD14. A nuclear run-on assay showed that CD14 transcription rates were significantly increased in hepatocytes from LPS-treated animals, indicating that the upregulation in CD14 mRNA levels observed in rat hepatocytes after LPS treatment is dependent, in part, on increased transcription. In vitro and in vivo experiments indicated that interleukin-1beta and/or tumor necrosis factor alpha participate in the upregulation of CD14 mRNA levels in hepatocytes. Our data indicate that hepatocytes express CD14 and that hepatocyte CD14 mRNA and protein levels increase rapidly during endotoxemia. Our observations also support the idea that soluble CD14 is an acute-phase protein and that hepatocytes could be a source for soluble CD14 production.
Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant
to endotoxin yet highly susceptible to Gram-negative infection. The codominantLps
d allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene
(Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous
for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces
the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.
CD14, a protein expressed on the surface of monocytes and neutrophils, is a major receptor for lipopolysaccharide (LPS). Studies
with normal and CD14-deficient macrophages show that responses to low concentrations of LPS require expression of CD14, whereas
responses to high concentrations of LPS are CD14-independent. Since LPS isolated from different bacterial species shows structural
variability, studies were performed to determine whether differences in LPS structure influence CD14-dependent and CD14-independent
responses. Studies with LPS purified from Escherichia coli, Salmonella abortus subspecies equi, Salmonella minnesota, Pseudomonas aeruginosa, Neisseria meningitidis, Bacteroides fragilis, and Rhodobacter sphaeroides show that the strongest CD14-dependent responses require a typical O-antigen, long carbohydrate chains, at least 6 acyl chains
in their lipid A, and 2-phosphorylated Kdo moieties; wild-type LPS lacking a typical O-antigen and containing short carbohydrate
chains and 2-phosphorylated Kdo moieties induces the strongest CD14- independent response.
To establish auto iris pigment epithelial (IPE) transplantation, we characterized the properties of IPE cells and the method of culture using auto serum. Monkey and human IPE cells were obtained and cultured in several conditions, using auto, mouse, rabbit, bovine, or human serum. Immunocytochemical study was performed to confirm that the cells were epithelial in origin. The proliferation rate of the IPE was also calculated from fresh human IPE cells, which were obtained during filtering glaucoma surgery. Proliferation rate was also compared to that of retinal pigment epithelial (RPE) cells. Reverse-transcriptase and polymerase chain reaction for melanogenesis was performed, and the amount of pigment in the IPE cells was also calculated. Mouse and rabbit sera were not effective for the monkey IPE cell culture. Conversely, the cells grew well in the medium with auto, bovine, or human serum. Human IPE cells grew exponentially by the described methods and reached to 60,000 cells after about 4-5 weeks. When we compared them by proliferation rate, IPE cells were less proliferative than RPE cells. The gene expression for melanogenesis and the amount of pigment in the IPE gradually decreased through successive passages. Transplantation has been tried for the treatment of age-related macular degeneration using RPE from fetus or from eye bank eyes. However, focal rejection may play an important role in the clinical results. The establishment of auto IPE cell transplantation may improve the problem of rejection. In the present study, we established auto IPE cell culture using auto serum. The cultured IPE cell showed pigment epithelial cell properties until around five passages in both human and monkey.
To improve monitoring of immunological and disease activity, we determined soluble markers of activity of the monocyte/macrophage system (sCD14) and the vascular endothelium (sE-selectin, sICAM-1) in patients with systemic lupus erythematosus (SLE) and primary Sjögren' syndrome (pSS) in comparison to patients with infections or sepsis.
Concentrations of sCD14, sICAM-1 and sE-selectin (soluble CD14, ICAM-1 and E-selectin, respectively) were measured in serum samples from patients with SLE and pSS, patients with sepsis, different infectious diseases and healthy controls using ELISA systems.
Elevated levels of sE-selectin and sICAM-1 were detected in patients with SLE as well as sepsis, in contrast to patients with a localized infection (SLE and sepsis, respectively, versus infection P < 0.001; Kruskal–Wallis test). Levels of sCD14 were persistently elevated in sera from patients with SLE, whereas these values decreased rapidly after effective therapy in patients with sepsis or infection. A continuous elevation of all of these three parameters was associated with a fatal outcome in patients with sepsis as well as in patients with SLE.
Combined elevation of sCD14, sICAM-1 and sE-selectin correlates with the prognosis in patients with active SLE and indicates a remarkable immune activation involving the monocyte/macrophage system and the endothelium comparable to an activation found only in patients with sepsis.
To study the infectious background of patients with a history of acute anterior uveitis (AAU) and healthy control subjects.
Sixty four patients with previous AAU and 64 sex and age matched controls were studied. Serum antibodies to Salmonellae, Yersiniae, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Campylobacter jejuni, and Borrelia burgdorferi were measured using enzyme linked immunosorbent assay (ELISA), and antibodies to Chlamydia trachomatis and Chlamydia pneumoniae by microimmunofluorescence test. Peripheral blood mononuclear cells (PBMCs), separated by density gradient centrifugation, were studied for Salmonella and Yersinia antigens by means of an immunofluorescence test, and for C pneumoniae DNA with a polymerase chain reaction (PCR).
Neither prevalence nor levels of single microbial antibodies studied differed between the patients and control subjects, or between subgroups of patients created on the basis of clinical characteristics. In logistic regression analysis, the high number of recurrences (>10) of AAU was independently related to the presence of single or multiple bacterial antibodies (p=0.04). None of the PBMC samples of the patients were positive for Yersinia or Salmonella antigens. C pneumoniae PCR was positive in a patient who was negative for C pneumoniae antibodies.
Although neither the prevalence nor the levels of single microbial antibodies studied differed between the patients and the controls, current data suggest that the presence of single or multiple antibodies in patients with many recurrences of AAU compared with patients with none or few recurrences may be a sign of repeated infections, antigen persistence, or raised innate immune responsiveness.
Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 angstroms resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.
To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate.
AqH from noninflammatory control subjects and patients with idiopathic, Fuchs' heterochromic cyclitis (FHC), and herpes-viral or Behçet's uveitis were analyzed for IL-1beta, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNFalpha, IFNgamma, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFbeta2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample.
Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFNgamma, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFbeta2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNFalpha, and IL-2.
Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.
Lipopolysaccharide (LPS) is one of the most powerful bacterial virulence factors in terms of proinflammatory properties and is likely to contribute to corneal bacterial keratitis. Better understanding of the spatial expression of the LPS receptor components at the tear-corneal interface might facilitate enhanced functions of the LPS receptor complex in ocular defense against Gram-negative infections.
The expression of LPS-binding protein (LBP), CD14, toll-like receptor (TLR)-4, and MD-2 in human lacrimal glands, reflex tears, and corneal epithelia was examined by ELISA, RT-PCR, Western blot analysis, and immunofluorescence. The release of proinflammatory cytokines after the activation of primary and immortalized corneal epithelial cells with LPS and human tears was measured by ELISA.
LBP and CD14 proteins were detected in reflex human tears. Human lacrimal glands and corneal epithelia expressed LBP, CD14, TLR4, and MD-2 mRNAs and proteins. In the corneal epithelium, LBP was mainly expressed by superficial and basal epithelial cells, whereas CD14, TLR4, and MD-2 expression were limited to the wing and basal epithelial cells. In a dose-dependant manner, tear CD14 and LBP mediated the secretion of interleukin (IL)-6 and IL-8 by corneal epithelia cells when challenged with LPS.
Tear CD14 and LBP complemented the LPS receptor complex expressed by the corneal epithelia to trigger an immune response in the presence of LPS. The complementation of these tear and corneal immune proteins could play an important role in LPS recognition and signaling and, therefore, could modulate ocular innate immunity.
It was previously demonstrated that toll-like receptor 4 (TLR4) is involved in species-specific human retinal pigment epithelial (HRPE) photoreceptor outer segment recognition and oxidant production. This study was performed to demonstrate the classical role of TLR4 in HRPE endotoxin (lipopolysaccharide; LPS) binding leading to HRPE proinflammatory cytokine secretion.
Cultured HRPE cells were examined for TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. HRPE cells labeled with fluorescent monoclonal antibodies (mAbs) to TLR4 and its associated adhesion molecule, CD14, were analyzed by real-time microscopy and resonance energy transfer (RET), determining associations of fluorescent LPS, TLR4, and CD14. LPS-induced HRPE secretion of interleukin (IL)-8 was measured with and without specific blocking mAb to TLR4 and CD14. HRPE TLR4 expression was measured after LPS exposure in the presence and absence of blocking antibodies to TLR4 and CD14.
All three HRPE cell lines demonstrated constitutive TLR4 expression by immunofluorescence, Western blot analysis, and RT-PCR. Examination of HRPE cells labeled with fluorescent mAb to TLR4, CD14, and LPS demonstrated RET among the three molecules, indicating that LPS-CD14 binding preceded LPS-TLR4 binding and the close association of CD14 and TLR4. LPS-induced IL-8 was significantly reduced (P < 0.05) in the presence of blocking mAb to TLR4 or CD14. Upregulation of HRPE TLR4 gene and protein expression occurred in response to LPS exposure and was inhibited by mAb to TLR4 or CD14.
HRPE TLR4 is a multifunctional molecule that participates in photoreceptor outer segment membrane recognition, oxidant production, LPS recognition, and cytokine production. These roles indicate potential involvement in retinal degenerative and inflammatory processes.
Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. There is no effective treatment for the most prevalent atrophic (dry) form of AMD. Atrophic AMD is triggered by abnormalities in the retinal pigment epithelium (RPE) that lies beneath the photoreceptor cells and normally provides critical metabolic support to these light-sensing cells. Secondary to RPE dysfunction, macular rods and cones degenerate leading to the irreversible loss of vision. Oxidative stress, formation of drusen, accumulation of lipofuscin, local inflammation and reactive gliosis represent the pathologic processes implicated in pathogenesis of atrophic AMD. This review discusses potential target areas for small-molecule and biologic intervention, which may lead to development of new therapeutic treatments for atrophic AMD.
Study of models of ocular autoimmunity and of autoimmune uveitis in humans has lead to a shift in the perceived nature of immune privilege from one based on anatomical isolation of the eye to a more dynamic, active process of immune tolerance. Using a variety of available models, the basis for this dynamic process of immune regulation is reviewed. The protective role of humoral immunity, the co-stimulatory function of B cells in EAU as well as the influence of cytokines within the inflammatory cascade are outlined. Modulation of the immune response and in particular the possible role of macrophages is explored. Within the current paradyme, a major effector cell is the CD4+ lymphocyte. Its maturation into a Th1 or Th2 phenotype process appears dependent on a number of exogenous factors, which while genetically determined can be manipulated prior to disease onset. Activation of CD4+ cells is dependent on presentation of immunoreactive peptide fragments. These fragments are well characterized in the Lewis rat for S-Ag and interphotoreceptor retinoid binding protein (IRBP). Mapping of the immunoreactivity to S-Ag has been recently completed in uveitis patients. An overlap with certain determinants identified in experimental models has been observed, in at least 2 disease entities. However, the response profile is not fixed in time and is subject to determinant spread. Future studies will be aimed at identifying with more detail immunologic triggers of inflammation in patients, and at better defining the interplay between effector and regulatory pathways both in the eye and in the systemic circulation.
It has been suggested that human iris pigment epithelial (IPE) cells isolated from iridectomized tissue could be used as autologous cells for transplantation into the subretinal space in diseases with dysfunctional retinal pigment epithelium (RPE). RPE cells synthesize a number of cytokines and their receptors which are important for its proper function. Nearly nothing is known about the capacity of IPE to synthesize cytokines or responding to them. To compare the mRNA expression of 36 cytokines or their receptors in cultured adult IPE cells and RPE cells we used semi-quantitative reverse transcription polymerase chain reactions (RT-PCR). Included in our assay were cytokines with known expression in RPE to get a broad basis for comparing IPE cells: basic fibroblast growth factor (bFGF or FGF-2), and one of its receptor (FGFR-1), epidermal growth factor (EGF), and its receptor EGF-R, transforming growth factor β(TGFβ), and its type III receptor TGFβ-R3, the platelet-derived growth factors and receptors (PDGF A, PDGF B, PDGF-Rα, PDGF-Rβ), tumor necrosis factor α(TNFα), and two receptors TNF-R1 and TNF-R2, insulin (INS) with receptor INS-R, insulin-like growth factors (IGF1, IGF2), and receptors (IGF1-R, IGF2-R), vascular endothelial growth factor (VEGF), and two receptors (VEGF-R1 or FLT-1 and VEGF-R2 or FLK-1), the receptor for VEGF-C: VEGF-R3 or FLK-4, interleukin 6 (IL6), and its receptor (IL6-R), nerve growth factor (NGF), interleukin 1α(IL1α), and a receptor (IL1-R). In addition, cytokines or their receptors not known to be expressed in RPE were included to widen our picture of cytokine gene expression in the eye: stem cell factor (SCF), its receptor (SCF-R), low-affinity nerve growth factor receptor p75 (p75(NGF-R), ciliary neutrothropic factor (CNTF), and its receptor (CNTF-R), glycoprotein 130 interleukin 6 transducer (gp130 (IL6-SD), leukemia inhibitory factor (LIF), and its receptor (LIF-R). Semi-quantitative expression data were obtained using series of fivefold dilutions of each cDNA and a fixed number of PCR cycles. The expression of RPE 65, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β2-microglobulin (B2MG) was used as a control for cellular origin, RNA quality and PCR conditions.With the exception of insulin and tumor necrosis factor αall other cytokines analysed and their receptors were expressed in both IPE and RPE cells, even though the levels varied. No qualitative or quantitative difference were observed in the mRNA expression level of 34 (94%) of the cytokines or receptors between IPE and RPE. In contrast, the mRNA expression level of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 [VEGF-RS (FLK-1)] was lower in IPE than in RPE cells. As an increased expression of VEGF in the RPE in maculae with age-related macular disease could be involved in its pathogenesis, a decreased expression of angiogenic growth factors in IPE cells could possibly be beneficial for the therapy of age-related maculopathy if indeed other tasks of non-functional RPE cells could be performed by IPE cells. The similarity of the mRNA expression pattern in 94% of the cytokines analyzed supports the assumption that IPE cells potentially can perform functions of RPE cells in the appropriate environment.
Inflammation is a major contributing factor to many blinding disorders including uveitis, diabetic retinopathy, and age-related macular degeneration. Here we examined the response of the retinal pigment epithelium (RPE) to physiological levels of lipopolysaccharide (LPS) to understand the role of this epithelium in inflammatory retinal conditions. Expression of a group of inflammatory mediators was identified by gene array analysis and confirmed by PCR and immunocytochemistry in primary human RPE cultures and ARPE19. The effects of LPS on the expression of these cytokines and RPE survival were examined by PCR, Luminex bead, and MTT assays. RPE cells express many cytokine receptors including IL-1R, -4R, -6R, -8RA, IFNAR1, IFNGR1/2 and secrete a range of pro- and anti-inflammatory cytokines including IL-4, -6, -8, -10, -17, IFN-γ, MCP-1, and VEGF. LPS increases IL-13RA1 and IFNAR1, and decreases IL-7R receptor expression. It also increases RPE secretion of IL-4, -6, -8, -10, IFN-γ and MCP-1, and is toxic to RPE cells at LC50 = 17.7 μg/ml. LPS toxicity is mediated by IL-6 and IL-8 through an autocrine feedback loop. Silencing IL-6R and IL-8RA gene expression by siRNA blocks death by their respective ligands or LPS. These findings imply that RPE cells are acutely sensitive to inflammatory stress and that over secretion of IL-6 and IL-8 by this epithelium during inflammatory stimulus may be an underlying factor in the progression of some retinal pathologies.
Acute anterior uveitis (AAU) may be associated with systemic infectious or inflammatory disease. We examined 92 patients with the first attack of acute anterior uveitis; all patients were free of any extraocular symptoms. A thorough clinical examination did not reveal any systemic underlying disease. In the course of microbiological examination, however, a high incidence of asymptomatic infection of the urethra and/or cervix with ureaplasma urealyticum, chlamydia trachomatis and mycoplasma hominis was found. Infections with ureaplasma were significantly more frequent in patients with AAU when compared with a sex- and age-matched control group. There was no statistically valid association of these infections with the HLA-B27 phenotype in the patients. The higher rate of urogenital infections in patients with AAU may reflect a higher rate of sexual promiscuity. Transmission of infectious agents seems to be one possible factor in the pathogenesis of AAU.
Luminol-enhanced chemiluminescence was used to determine the effect of soluble CD14 (sCD14) on the endotoxin-inducible generation of reactive oxygen species in human monocytes. It was necessary to mediate lipopolysaccharide (LPS) monocyte-activating capability by serum factors (LPS-binding proteins). sCD14 reduced LPS-inducible monocyte activation in a dose-dependent manner, even in the case of CD14- monocytes, obtained from a patient with paroxysmal nocturnal haemoglobinuria. These monocytes could be activated by opsonized LPS via other receptors. Using anti-mouse Ig-coated microbeads, it was demonstrated in FACS analysis that sCD14 mediates the binding of a mouse monoclonal anti-CD14 antibody (RoMo 1) to a complex of LPS/FITC (fluoroisothiocyanate) and a LPS-binding protein. The release of sCD14 from cultured monocytes was measured using LPS, TNF alpha (tumour necrosis factor), IL1, 4 and 6 (interleukin-1, -4 and -6) and IFN gamma (interferon-gamma) as stimulators. Addition of LPS and TNF alpha led to a dose-dependent increase in sCD14-levels in the culture supernatant, whereas IL1, IL6 and IFN gamma had no significant effect. IL4 dose-dependently depressed spontaneous sCD14 release. It is possible that elevated sCD14-serum levels in polytraumatized patients indicate a natural protective mechanism against excessive monocyte mediator production. Therefore, sCD14 may be a new therapeutic concept in endotoxic shock prevention.
A coherent view of the role of cytokines in inflammatory eye disease is emerging as a result of studies both in man and experimental animals. Cytokines have been demonstrated in ocular tissue obtained from patients with intraocular inflammation (uveitis) (gamma interferon, IL-2) and have been shown to induce inflammation in experimental animals after intraocular injection [(IL-1, IL-6, IL-8, tumour necrosis factor (TNF), granulocyte macrophage-colony stimulating factor (GM-CSF)]. Several unique features of the immunology of the eye such as the immunosuppression associated with anterior chamber associated immune deviation (ACAID) may be due to the effects of cytokines. Similarly, common complications of ocular inflammation such as glaucoma, keratic precipitates, retinal (macular) oedema and neovascularization may be mediated by cytokines. Understanding of the role of cytokines in inflammatory eye disease has the potential to lead to the development of therapies to abrogate the effects of these important mediators of the inflammatory response.
Acute anterior uveitis is a relatively common disease of uncertain aetiology. Its association with a wide variety of systemic disorders including ankylosing spondylitis, Reiter's syndrome, Behçet's disease, juvenile rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease and sarcoidosis and the prevalence of the HLA antigen B27 (ref. 1) in cases without associated disease have been noted but not explained. In general, animal models for uveitis have not clarified the mechanism of human disease. Most require direct intravitreal injection2 or repeated immunizations with retinal or uveal antigens3. Uveitis does occur in adjuvant arthritis, a syndrome that can be produced in rats by the systemic injection of pepti-doglycan from the cell wall of a variety of Gram-positive bacteria4. The disease, which was initially described following the injection of mycobacteria in mineral oil, has many similarities to Reiter's syndrome5, including the development of eye pathology in as many as 40% of the animals6. As Reiter's syndrome is associated with enteric Gram-negative infections such as Shigella, Salmonella and Yersinia, we sought to produce an arthritis in rats by injecting killed Gram-negative bacteria. Although we have not observed clinical arthritis in these animals, careful histological study has shown the near-universal appearance of uveitis. The studies described below indicate that the eye disease is due to a portion of the bacterial cell wall known as endotoxin or lipopolysaccharide (LPS).
CD14 functions as a cell surface receptor for LPS in the activation of monocytes/macrophages and neutrophils by endotoxin. To assess the utility of soluble forms of the CD14 receptor as a possible therapeutic for endotoxin shock, we have produced recombinant human soluble CD14 using a baculovirus expression system. We find that the recombinant protein is not only expressed on the surface of the insect cells as a glycosyl phosphatidylinositol (GPI)-anchored protein, but is also released into the culture medium as a soluble form that lacks the GPI anchor. Functional analyses of recombinant human soluble CD14 show that it binds specifically to LPS and can inhibit the LPS-induced release of TNF-alpha by macrophages and mononuclear cells as well as by cells in whole human blood when used at concentrations of approximately 70 micrograms/ml. Thus, soluble CD14 may be useful as an adjunct in the treatment of endotoxin shock.
The retinal pigment epithelium (RPE) plays a critical role in the development and maintenance of adjacent photoreceptors in the vertebrate retina. This study describes the development and characterization of ARPE-19, a spontaneously arising human RPE cell line with normal karyology which forms polarized epithelial monolayers on porous filter supports. The cell line was established by selective trypsinization of a primary RPE culture resulting in a uniform population of highly epithelial cells which exhibit a strong growth potential. To determine the extent of biochemical differentiation, the expression of the RPE-specific markers CRALBP and RPE65 was examined by Northern analysis. A single 1.6 kb CRALBP mRNA transcript and a single 2.8 kb RPE65 transcript were detected in samples of ARPE-19 total mRNA. The expression of CRALBP protein in ARPE-19 cell lysate was detected by Western blot analysis and immunocytochemistry was used to detect CRALBP throughout the cytoplasm of most, but not all, cells in confluent cultures. The essential criteria for monolayer formation were determined experimentally and it was found that ARPE-19 cells exhibit morphological polarization when plated on laminin-coated Transwell-COL filters in medium with a low serum content. The time course of tight-junction formation was determined by recording the transepithelial resistance of monolayers and reached a maximum of 50-100 omega cm2 after 4 weeks of culture. Barrier properties of ARPE-19 monolayers were evaluated by measuring the flux of 3H-inulin from the apical to the basolateral compartment of cell culture chambers. Finally, ARPE-19 clonal sublines were generated by serial dilution in an attempt to produce a subline with a high transepithelial resistance (TER). The morphology of the sublines was variable and the cloned cells exhibited a tendency to senesce in culture, confirming that this cell line is not transformed. No subline monolayers developed a TER greater than those recorded for the parent cells. Our results demonstrate that ARPE-19 has structural and functional properties characteristic of RPE cells in vivo and suggest that this cell line will be valuable for in vitro studies of retinal pigment epithelium physiology.
The purpose of this study is to measure soluble CD14 (sCD14) levels in sera from newborn with sepsis, to compare it with other markers, and to study its evolution in Gram-negative and Gram-positive sepsis. Forty normal newborns were included (26 were full term and 14 were preterm infants), 20 babies had a positive blood culture (11 Gram-positive and 9 Gram-negative) and 16 cases were suspected of having sepsis based on clinical and laboratory findings, but a negative blood culture. Interleukin-6 (IL-6), sCD14, and tumour necrosis factor-alpha (TNF alpha) were measured by enzyme immunoassay, and fibronectin (FN) and C-reactive protein (CRP) by radial immunodiffusion. Neonates with a positive blood culture had increased levels of sCD14 (3.20 +/- 1.26 micrograms ml-1, p < 0.001), CRP (69 +/- 46 micrograms ml-1, p < 0.001) and IL-6 (134 +/- 150 pg ml-1, p < 0.001), and decreased values of FN (12.3 +/- 6.6 mg ml-1, p < 0.001). TNF alpha levels were also high (160 +/- 37 pg ml-1), but this increase was not statistically significant. Newborn infants suspected of having sepsis but a negative blood culture had similar but milder abnormalities. Soluble CD14 levels correlated with CRP values; however, there was no correlation between sCD14, TNF alpha and IL-6. Neonates with sepsis by Gram-positive bacteria had lower sCD14 levels than patients with Gram-negative sepsis (2.63 +/- 1.2 versus 4.04 +/- 1.0 micrograms ml-1, p < 0.05). In conclusion, the sCD14 level is increased in newborn infants with sepsis, and this is higher in infections by Gram-negative bacteria, suggesting a different contribution of monocyte and macrophage cells. In contrast, IL-6, TNF alpha, CRP and FN values are similar in infections by Gram-positive and Gram-negative bacteria.
The aim of this study was to define the expression of chemoattractant cytokines (chemokines) in human aqueous humor, obtained from patients with idiopathic acute anterior uveitis (AU). The chemokines assayed included macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha and -1 beta), monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interferon-inducible protein-10 (IP-10), and regulated on activation, normal T-expressed and secreted (RANTES).
We studied fifteen patients (7 females) with idiopathic acute AU, at various stages of disease activity, and two control subjects undergoing elective cataract extraction. Aqueous humor was collected under aseptic conditions, after obtaining informed consent. Chemokine concentrations were measured using specific ELISA. Correlation was sought between chemokine concentrations and disease activity, evaluated by slit lamp biomicroscopy and graded using a standardized scale of disease severity.
IL-8 was detected (35.9 +/- 13.6, mean +/- SE) in three of seven subjects in active, untreated stages of AU (clinical score 2-4), and it was undetectable in subjects sampled in the quiescent phase of the disease. IP-10 had a mean concentration of 40.6 ng/ml +/- 20.9 in the active group (N = 7), declining to 0.8 ng/ml +/- 0.3 in the samples from patients with inactive disease (N = 7, P = 0.001). Similarly, substantial expression of MCP-1 was noted, with a maximum concentration of 145 ng/ml, in acute (active) AU (N = 6), (26.7 +/- 19.7), falling to undetectable levels in those with inactive disease, and in control subjects (P = 0.001). MIP-1 beta (N = 7), (3.4 +/- 1.5, P = 0.001) and RANTES (N = 7, 8.8 +/- 4.2) levels were significantly increased in acute disease (P = 0.001) and related to the activity of the disease, although the concentrations were not as high as MCP-1, IP-10 and IL-8. IP-10, RANTES and MIP-1 beta were detected at low concentrations in the aqueous humor of the control subjects.
This is the first study of chemokine concentrations in the aqueous humor of patients with acute anterior uveitis. The concentration of chemokines: IL-8, IP-10, MCP-1, RANTES and MIP-1 beta were significantly increased during the active stages of AU, and correlated with the clinical severity of the disease. These chemoattractant cytokines probably play a critical role in leucocyte recruitment in acute AU.
FHC and IAU are two forms of anterior uveitis which are localized to the eyes with no evidence of systemic involvement. However, FHC has distinct clinical features and differs from IAU in that the inflammation is low grade, steroid non-responsive, and has a less aggressive clinical course. To try to dissect the mechanism for this difference the phenotypes of the cells in the AH and blood (PB) and the cytokines present in the AH in patients with FHC and IAU were compared. Three-colour flow cytometry was performed on the cells isolated from the AH and PB. Percentage of cells bearing the following markers were determined: CD3, CD4, CD8, CD4/CD25, CD8/CD25, CD19 and CD14. The cytokines IL-4, IL-10, IL-12 and interferon-gamma (IFN-gamma) were assayed by ELISA. In both groups T cell numbers were higher in the AH than PB, although the distribution of T cell subsets in PB was similar. In the AH, CD8+ T cell numbers were higher in FHC than in IAU (P = 0.003), whilst CD4+ numbers were higher in IAU than FHC (P = 0.01). AH cytokine profiles were different in the two groups: IFN-gamma levels were higher and IL-12 levels lower in the FHC group than IAU (P = 0.02), whilst IL-10 levels tended to be higher in the FHC group (P = 0.5). We suggest that different local mechanisms governing the balance of T cell/cytokine-mediated inflammation in the anterior segment may underlie clinical differences such as chronicity and response to steroids in these disorders.
To identify the cellular immune processes underlying intra-ocular inflammation, aqueous humour was obtained at cataract surgery from 22 patients with clinically inactive uveitis and 24 patients with age-related cataract. mRNA expression for the cytokines IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta); T cell subsets CD3, CD4, CD8; monocytes and macrophages (CD14); and B cells (CD19) was measured using reverse transcriptase-polymerase chain reaction (RT-PCR) and radiometric analysis. The majority of uveitis patients demonstrated a T cell-mediated inflammatory response, predominately involving a Th1-like cytokine profile with expression of IL-2 and IFN-gamma in 16/22 and 18/22 samples, respectively. These cytokines were present in only a small number of patients with age-related cataract. This Th1-like polarization was supported by an increased expression of CD8 in a number of patients. IL-1beta was expressed in only six uveitic eyes. Only four patients expressed either IL-4 or IL-10 and no patient expressed both. TGF-beta mRNA could be detected in 18/22 uveitis patients and 15/24 controls. IL-12, the paradigmatic Th1-inducing cytokine, was absent in all samples but CD14 was expressed in the majority of patients and controls. CD19 could not be detected in any sample. The cellular infiltrate in the uveitic eyes showed clear evidence of low IL-1 and absent IL-12 expression despite a Th1-like profile and high expression of macrophages. This strongly suggests that the systemic immunosuppressive therapy used prior to surgery in some patients and/or the chronicity of the uveitis had actively suppressed/switched off macrophage function, leading to resolution of T cell activity.
Lipopolysaccharide (LPS, or endotoxin), is a major constituent of the outer membrane of Gram-negative bacteria. Bacteria express either smooth LPS, which is composed of O-antigen (O-Ag), complete core oligosaccharides, and the lipid A, or rough LPS which lack O-Ag but possess lipid A and progressively shorter core oligosaccharides. CD14 has been described as the receptor for complexes of LPS with LPS-binding protein (LBP). Using flow cytometry we have compared the binding of Salmonella minnesota rough LPS (ReLPS) and Escherichia coli smooth LPS labelled with fluorescein isothiocyanate (FITC-LPS) to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO), to MonoMac 6 cells and to endothelial cells. Our results showed that both forms of LPS display the same binding characteristics, and that the binding of FITC-LPS to cells was both CD14- and LBP-dependent for LPS concentrations up to 100 ng.mL-1. At LPS concentrations higher than 100 ng.mL-1 we observed CD14/LBP-independent binding. CD14/LBP-dependent binding was dose dependent, saturable, and enhanced in the presence of human pooled serum (HPS), and the monoclonal anti-CD14 antibody (MY4) or unlabelled LPS could outcompete it.
Vision is dependent on proper function of several intraocular structures. Immune responses to eliminate invading pathogens from the eye may threat vision by causing damage to these structures. Therefore, immunological defence of the eye should be carefully balanced between efficacy and maintenance of functional integrity. The eye is equipped with several regulatory mechanisms to prevent certain immune and inflammatory responses and is, therefore, regarded as an immune privileged site. The retinal pigment epithelium (RPE) contributes to the immune privileged status of the eye as part of the blood-eye barrier and by the secretion of immunosuppressive factors inside the eye. RPE cells, however, may also play an important role in the development of immune and inflammatory responses in the posterior part of the eye. During the last decade it has become clear that RPE cells are highly sensitive to a variety of inflammatory cytokines. Under inflammatory conditions, RPE cells produce a myriad of cytokines that may activate the resident ocular cells or attract and activate leukocytes. Cytokine stimulation of RPE cells causes profound effects, including nitric oxide secretion, cell surface expression of MHC class II and adhesion molecules and abrogation of barrier function. This article provides a comprehensive review of the literature concerning RPE cells and cytokines.
A number of studies have shown that transplantation of retinal pigment epithelial (RPE) cells to the subretinal space offers a promising treatment modality for retinal degenerative diseases. However, it is necessary to transplant autologous cells to avoid rejection; unfortunately, obtaining autologous RPE cells necessitates such traumatic surgical intervention as to make this approach irrelevant. It has been hypothesized that iris pigment epithelial (IPE) cells may be a possible substitute for RPE cells for transplantation into the subretinal space. The iris pigment epithelium, which has the same embryonic origin as retinal pigment epithelium, has not received much attention from visual scientists. Even though it forms a highly specialized tissue, it is not clear whether the iris pigment epithelium contributes critical functions to the health of the visual system. In vivo the IPE does not appear to have any of the functions characteristic of RPE; however, in vitro cultured IPE cells do acquire functions, such as specific phagocytosis of rod outer segments, that are characteristic of RPE cells, and have been shown to have the potential to carry out many functions characteristic of RPE cells, e.g., retinol metabolism. This review outlines the development and cellular functions of the IPE with special emphasis on the modulation of those functions that can allow the IPE cells to be transplanted to the subretinal space where they appear to acquire differentiated properties of retinal pigment epithelium (RPE).
The kinetics of various cytokines in the eye plays a critical role in endotoxin-induced uveitis (EIU). This study examined the cytokine kinetics and susceptibility of EIU in four mice strains.
Four strains of TLR-4 or Toll-like receptor-4 (Lps, lipopolysaccharide-susceptible) gene-positive mice (C3H/HeN of H-2(k), C57/B6 of H-2(b), Balb/C of H-2(d), and 129/J of H-2(b)) were injected subcutaneously with either lipopolysaccharide (LPS) in phosphate-buffered saline (PBS) or PBS alone in two repeated experiments. Mice were sacrificed 1, 3, 6, 24 (1 d), 72 (3 d), 120 (5 d), or 168 (7 d) hours after LPS injection. Ocular histology and reverse transcriptase-polymerase chain reaction (RT-PCR) to detect ocular interleukin-1 alpha (IL-1 alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA were performed. Serum IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha levels were measured using enzyme-linked immunosorbent assay (ELISA).
No ocular inflammation was present in any mice within six hours after LPS injection. Only the C3H/HeN mice developed a biphasic ocular inflammatory response (1 d and 5 d), during which all proinflammatory cytokine messages were expressed. In the other three strains with minimal (129/J and Balb/C) to mild (C57/B6) EIU that peaked at 1 d, IL-6 mRNA was barely detectable in C57/B6 and Balb/C; GM-CSF mRNA was also present in C57/B6. Serum IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha were high in all EIU mice within six hours after LPS injection. Control mice did not develop uveitis or measurable cytokine messages.
In the most susceptible strain, C3H/HeN, EIU was biphasic and correlated to multiple proinflammatory cytokines released in the eye. The less susceptible mice strains exhibited a monophasic response to LPS that may result from no cytokine cascade.
To determine the presence of systemic inflammation and innate immune responsiveness of patients with a history of acute anterior uveitis but no signs of ocular inflammation at the time of recruitment.
Tumour necrosis factor alpha (TNF-alpha) production in response to bacterial lipopolysaccharide (LPS) was studied using whole blood culture assay; levels of TNF-alpha in culture supernatants, and soluble interleukin 2 receptor (sIL-2R) in serum were determined by chemiluminescent immunoassay (Immulite); monocyte surface expression of CD11b, CD14, and CD16 and the proportion of monocyte subsets CD14(bright)CD16(-) and CD14(dim)CD16(+) were studied with three colour whole blood flow cytometry; and serum C reactive protein (CRP) levels were determined using immunonephelometric high sensitivity CRP assay.
The CRP level (median, interquartile range) was significantly higher in 56 patients with previous uveitis than in 37 controls (1.59 (0.63 to 3.47) microg/ml v 0.81 (0.32 to 2.09) microg/ml; p=0.008). The TNF-alpha concentration of the culture media per 10(5) monocytes was significantly higher in the patient group than in the control group in the presence of LPS 10 ng/ml (1473 (1193 to 2024) pg/ml v 1320 (935 to 1555) pg/ml; p=0.012) and LPS 1000 ng/ml (3280 (2709 to 4418) pg/ml v 2910 (2313 to 3358) pg/ml; p=0.011). The background TNF-alpha release into the culture media was low in both groups. CD14 expression of CD14(bright)CD16(-) monocytes, defined as antibody binding capacity (ABC), was similar for the patients and controls (22,839 (21,038 to 26,020) ABC v 21,657 (19,854 to 25,646) ABC).
Patients with previous acute anterior uveitis show high innate immune responsiveness that may play a part in the development of ocular inflammation.
Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. We show here that MD-2(-/-) mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection. We found that in MD-2(-/-) embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4.
CD14 is the primary receptor for lipopolysaccharide (LPS)that plays important roles in host defense and subserves other host-related biological functions. We previously identified CD14 on cultured human retinal pigment epithelial (HRPE) cells using immunocytochemical techniques. In this study, we investigated immunoreactive HRPE CD14 expression by immunohistochemically staining HRPE cells and HRPE cells in sections of human eyes with anti-CD14 monoclonal antibodies (mAb). Constitutive HRPE gene and protein expression were confirmed by semiquantitative PCR and western blotting. ELISA for cell-associated and secreted (s) HRPE CD14 revealed that specific digestion by phosphoinositol-specific phospholipase C (PI-PLC) significantly reduced (P<0.01) cell-associated HRPE CD14 which was not modulated by LPS or gamma-IFN. ELISA of the conditioned media (CM) of HRPE cells treated with PI-PLC contained significantly more (P<0.001) sCD14, but sCD14 was not modulated by LPS or gamma-IFN. FACS analysis confirmed HRPE cell surface CD14. To show functional CD14, fluorescently-labelled LPS and CD14 were demonstrated to show significant co-localization on live, cultured HRPE cells in close proximity (<7A) as demonstrated by resonance energy transfer of the fluorescent ligands (P<0.0001). Significant inhibition (P<0.001) of LPS-induced IL-8 secretion, as measured by ELISA, occurred in the presence of function blocking anti-CD14 mAb. Significant inhibition of LPS-induced HRPE IL-8 secretion by PKC, PTK, PI3 kinase, and p38 kinase inhibitors indicated cell mediators responsible for LPS-induced HRPE chemokine secretion. This study demonstrates that HRPE cells express functional CD14 in vitro and in situ along at the outer blood-retina barrier.
For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.
To determine whether there are differences in the immunopathogenesis of different endogenous uveitis syndromes, the phenotypic characteristics of immune cells were analysed among patients with endogenous uveitis. The aetiology of the uveitis included idiopathic recurrent acute anterior uveitis (18 patients), idiopathic intermediate uveitis (13 patients), Behçet's uveitis (17 patients), Vogt-Koyanagi-Harada syndrome (7 patients), and so on. Flow cytometric analysis was performed using immune cells of the aqueous humor and the peripheral blood during the active phase of intraocular inflammation, and monoclonal antibodies to CD3, CD4, CD8, CD14, CD19, CD56, TCR gammadelta, pan TCR alphabeta and Valpha24. CD8+ T cells were predominant in the aqueous humor of the patients with Behçet's uveitis, whereas CD4+ T cells were mainly found in the aqueous humor of patients other than those with Behçet's uveitis. The number of NKT (CD3+CD56+) cells was significantly higher both in the aqueous humor and the peripheral blood of the patients with Behçet's uveitis compared with the other groups (P < 0.05). CD8+CD56+ cells were the predominant subtype of the increased NKT cells in patients with Behçet's uveitis. In addition, intraocular infiltration of CD14+ cells significantly differed among the uveitis patients (P < 0.05). These results suggest that the immunopathogenesis of endogenous uveitis can vary between syndromes, and that CD8+CD56+ NKT cells may play an important role in the immunopathogenesis of Behçet's uveitis.
We investigated the expression of the functional endotoxin receptor proteins Toll-like receptor-4 and CD14 in human eyes. Toll-like receptor-4 and CD14 proteins were detected by immunohistochemical analysis of sections of whole human eyes embedded in paraffin with monoclonal antibodies against human toll-like receptor-4 (HTA-125), human CD14 (RPA-M1), or as a control, an irrelevant mouse IgG1k (MOPC-21). Incubation of explants with a neutralizing anti-toll-like receptor-4 monoclonal antibody was used to determine if lipopolysaccharide stimulation of tumor necrosis factor or interleukin-6 secretion was dependent on Toll-like receptor-4 activity. Reverse transcription-polymerase chain reaction was used to detect mRNAs for toll-like receptor-4, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8, 3 hr after stimulation of cultured iris microvascular endothelial cells. By immunohistochemistry, human ciliary body non-pigmented epithelial cells showed strong expression of the endotoxin receptor proteins, toll-like receptor-4 and CD14. Toll-like receptor-4 antibodies significantly inhibited lipopolysaccharide-stimulated tumor necrosis factor secretion by the ciliary body. Toll-like receptor-4 mRNA was constitutively expressed in iris endothelial cells and slightly down-regulated by endotoxin. mRNA levels for tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 were all increased by endotoxin treatment. This is the first report that shows intraocular (ciliary body and iris) expression of toll-like receptor-4, other than in cornea. Our results show that the ciliary body also expresses CD14, which is anatomically colocalized with toll-like receptor-4. This suggests a potential interaction between both molecules during endotoxin activation of ciliary body cells. The juxtaposition of toll-like receptor-4 and CD14 in the anterior uveal tract helps to explain the sensitivity of the iris/ciliary body to bacterial endotoxin as seen in the standard animal model of endotoxin-induced uveitis.
Acute anterior uveitis is the most common form of uveitis. HLA-B27-associated acute anterior uveitis is a distinct clinical entity that has wide-ranging medical significance due to its ocular, systemic, immunologic, and genetic features. The association between HLA-B27 and the spectrum of HLA-B27-associated inflammatory diseases remains one of the strongest HLA-disease associations known to date. This review examines acute anterior uveitis with particular focus on HLA-B27-associated acute anterior uveitis, including the epidemiology, immunopathology, association with HLA-B27 and its subtypes, clinical features, complications, prognosis, and potential new therapies such as anti-TNFalpha therapy and oral HLA-B27-peptide tolerance. There have been substantial recent advances in both clinical and basic scientific research in this field, including studies of the various animal models of acute anterior uveitis and the HLA-B27 transgenic animals, and these are summarized in this review. To the ophthalmologist, HLA-B27-associated acute anterior uveitis is an important clinical entity that is common, afflicts relatively young patients in their most productive years, and is associated with significant ocular morbidity due to its typically recurrent attacks of inflammation and its potentially vision-threatening ocular complications. Furthermore, to the ophthalmologist and the internist, HLA-B27-associated acute anterior uveitis is also of systemic importance due to its significant association with extraocular inflammatory diseases.
CD14 is a pattern recognition receptor; its important role in innate immunity is reviewed here. Since its discovery and subsequent classification at the first leucocyte typing workshop in 1982, CD14 has been thought of as a leucocyte differentiation antigen. However, it has become clear that CD14 is also expressed by many non-myeloid cells, and the evidence for this is presented. The possible role of the presence of low copy number CD14 on non-myeloid cells is discussed. It is time to acknowledge CD14 as an ubiquitous molecule and abandon the position that it is expressed by myeloid cells alone.
To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE.
ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays.
78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells.
While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.
Microbial agents have an important role in the pathogenesis of various inflammatory eye diseases, such as uveitis and keratitis. Microbial infections of the eye such as microbial keratitis, ocular onchocerciasis, bacterial endophthalmitis, viral retinitis, and other infectious uveitis are unfortunately common. In addition, microbial agents have been implicated in the pathogenesis of "non-infectious" immune mediated diseases such as HLA-B27 associated acute anterior uveitis. Toll-like receptors (TLR) are a family of pattern recognition receptors that initiates rapid host innate immune response to microbial components known as pathogen associated molecular patterns, which are unique to a given class of microbes, such as lipopolysaccharide of Gram negative bacteria. Recent in vitro and in vivo studies have demonstrated the expression and function of TLRs in the eye, with significant implications for better understanding of ocular immunity and the pathogenesis of inflammatory eye diseases affecting the cornea, uvea, and retina.
ARPE19 is a spontaneously immortalized cell line of human retinal pigment epithelium (RPE) that is used widely to draw inferences about the behavior of adult human RPE (ahRPE). We used DNA microarray analysis to compare the gene expression profiles of these two cell types.
Second-passage cultured ahRPE from four human donors (age range 48-82 years) and ARPE19 cultured to confluence in five dishes were used for this DNA microarray study. Total RNA was isolated and first- and second-strand complimentary DNA was synthesized using standard techniques. Biotin-labeled antisense complimentary RNA was produced by an in vitro transcription reaction. Target hybridization, washing, staining, and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Microarray data were normalized and statistical techniques were used to determine the presence or absence of expression of individual genes within ARPE19 and ahRPE, and their relative expression levels.
Hierarchic clustering analysis demonstrated that the gene expression profile of ahRPE and ARPE19 samples cluster into two distinct groups with no discernable overlap. The expression of 5,634+/-65 gene probes (out of 12,600 on microarray Human U95Av2 chip) was detected in ARPE19 cells compared to 5,580+/-84 genes in ahRPE cells from four human donor eyes. Thirty-five genes are expressed exclusively in ahRPE and nine genes exclusively in ARPE19 cells. Fifty additional genes have a threefold increase and 40 genes have a threefold decrease in expression level in ahRPE compared to ARPE19. There was no clear difference in the global expression level of genes known to be related to phagocytosis, angiogenesis, or apoptosis.
There are significant differences in the gene expression profile of ahRPE compared to ARPE19, and with some genes exclusively being expressed in one group and other genes being upregulated or downregulated by threefold. Caution should be exercised when generalizing results obtained from ARPE19 to the behavior of ahRPE.
Functional research of retinal pigment epithelium (RPE) most often relies on utilization of RPE-derived cell lines in vitro. However, no studies about similarities and differences of the respective cell lines exist so far. Thus, we here analyze the proteome of the most popular RPE cell lines: ARPE-19 and hTERT and compare their constitutive and de novo synthesized protein expression profiles to human early passage retinal pigment epithelial cells (epRPE) by 2-D electrophoresis and MALDI-TOF peptide mass fingerprinting. In all three cell lines the baseline protein expression pattern corresponded well to the de novo synthesized cellular proteome. However, comparison of the protein profile of epRPE cells with that of hTERT-RPE cells revealed a higher abundance of proteins related to cell migration, adhesion, and extracellular matrix formation, paralleled by a down-regulation of proteins attributed to cell polarization, and showed an altered expression of detoxification enzymes in hTERT-RPE. ARPE-19 cells, however, exhibited a higher abundance of components of the microtubule cytoskeleton and differences in expression of proteins related to proliferation and cell death. epRPE cells, hTERT-RPE, and ARPE-19 therefore may respond differently with respect to certain functional properties, a finding that should prove valuable for future in vitro studies.
To investigate the global cytokine and chemokine expression pattern in the aqueous humor of uveitis patients and relate them to clinical features.
Cross-sectional study.
In 31 aqueous humor samples from uveitis patients, the concentration of mediators was measured by a multiplex immunoassay. Eleven control samples were included.
Uveitis samples had higher levels of interleukin-6, interleukin-8, soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule (sVCAM), and interferon-inducible protein-10 (IP-10) than nonuveitis controls. Active uveitis samples had higher levels of interleukin-6, interleukin-10, interferon-gamma, sVCAM, regulated on activation, normal T cell expressed and secreted (RANTES), and IP-10 than quiescent uveitis samples. Infectious uveitis was associated with higher levels of interleukin-10 than noninfectious uveitis (P < .03 for all subgroups). No significant differences were found between cystoid macular edema (CME) and non-CME samples.
Elevated levels of specific mediators were found in active and in infectious uveitis, but not in CME. Mediator profiles might lead to a better understanding of the pathogenesis of uveitis.
The ocular microenvironment is immunosuppressive and anti-inflammatory. Pigment epithelial (PE) cells isolated from the eye possess a new property of suppressing T cell receptor-dependent activation of T cells in vitro. This property depends on their capacity to produce cell-surface and soluble inhibitory molecules. The iris pigment epithelia (IPE) do so through direct cell-to-cell contact with naïve T cells, and this suppressive contact is mediated by interactions between B7 and membrane-bound TGFbeta that are expressed constitutively on IPE. We have now examined whether other ocular PE cells, e.g., retinal pigment epithelia (RPE) and ciliary body pigment epithelia (CBPE), have a similar suppressive property by a similar process. We have found that RPE and CBPE significantly suppress the activation of bystander T cells via soluble inhibitory factors. RPE and CBPE secrete different soluble inhibitory factors including TGFbeta1 and TGFbeta2. Although IPE cells suppress the activation of bystander T cells by membrane-bound TGFbeta, the RPE and CBPE do so by soluble forms of active TGFbeta through mechanisms independent of cell contact. These ocular PE cells are capable modifying T cell function by enhancing production of regulatory cytokines including TGFbeta. We propose that this mechanism of suppression via TGFbeta ensures that soluble active TGFbeta is released into the ocular microenvironment in order to create the immune privilege of the posterior segment of the eye.
Despite significant progress in understanding the origin of soluble CD14 (sCD14), its physiological function remains largely unknown. Recent research has produced contradictory observations suggesting that sCD14 may have either beneficial or detrimental properties in protection against LPS-induced endotoxin shock. To resolve this controversy and to establish a mouse model suitable for elucidation of the functions of human CD14 (hCD14) in vivo, we generated several lines of transgenic mice bearing different copy numbers of the hCd14 transgene on a murine Cd14-/- background. The hCD14 was entirely capable of complementing loss of mouse CD14 to mediate cellular responses to LPS. Serum levels of sCD14 in a founder with multiple copies of the transgene were several times higher than in transgenic animals with a single copy of Cd14. Furthermore, mice with high levels of hCD14 were hypo-responsive to LPS and survived a lethal dose of LPS. Further inquiry into the mechanism of the hypo-response to LPS revealed that protection is associated with the higher amounts of circulating LPS. Most of this circulating LPS can be immunoprecipitated with anti-CD14 antibodies. These results suggest that sCD14 blocks circulating LPS by limiting the amount of monocyte-bound LPS and thus reduces inflammatory responses.
The iris plays a key role in visual function. It regulates the amount of light entering the eye and falling on the retina and also operates in focal adjustment of closer objects. The iris is involved in circulation of the aqueous humor and hence functions in regulation of intraocular pressure. Intriguingly, iris pigmented cells possess the ability to transdifferentiate into different ocular cell types of retinal pigmented epithelium, photoreceptors and lens cells. Thus, the iris is considered a potential source for cell-replacement therapies. During embryogenesis, the iris arises from both the optic cup and the periocular mesenchyme. Its interesting mode of development includes specification of the peripheral optic cup to a non-neuronal fate, migration of cells from the surrounding periocular mesenchyme and an atypical formation of smooth muscles from the neuroectoderm. This manner of development raises some interesting general topics concerning the early patterning of the neuroectoderm, the specification and differentiation of diverse cell types and the interactions between intrinsic and extrinsic factors in the process of organogenesis. In this review, we discuss iris anatomy and development, describe major pathologies of the iris and their molecular etiology and finally summarize the recent findings on genes and signaling pathways that are involved in iris development.
Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD-2 and
Toll-like receptor 4. Crystal structures of human MD-2 and its complex with the antiendotoxic tetra-acylated lipid A core
of LPS have been determined at 2.0 and 2.2 angstrom resolutions, respectively. MD-2 shows a deep hydrophobic cavity sandwiched
by two β sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located
at the entrance to the cavity. These structures suggest that MD-2 plays a principal role in endotoxin recognition and provide
a basis for antiseptic drug development.