[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.
Veterinary Immunology and Immunopathology 12/2011; 144(3-4):247-54. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biological specimens are often contaminated with bacteria-derived products such as LPS or lipoproteins (LP), which trigger unwanted inflammatory responses in hosts. Whereas a contamination by LPS can be determined by various test systems, a contamination by LP can as yet not be determined. TLR4 and TLR2 are key components of the LPS and the LP receptor complex, respectively. It was tested in this study whether HEK293 cell stably transfected with bovine TLR2 have the ability to react to low concentrations of diacylated and triacylated synthetic LP. The stable cell lines we present here recognize low concentrations of synthetic LP resembling LP of different bacteria. Therefore, these cells are suitable to detect low contaminations present in probes. For example, HEK293 cells stably transfected with bovine TLR2 recognized an egg albumin preparation as contaminated, as evidenced by copious production of IL-8. In contrast, these cells did not respond to a highly purified human serum albumin (HSA) preparation used in the clinic but responded to HSA containing small amounts of diacylated synthetic LP. The TLR4 ligand LPS is often said to activate TLR2. Here we present evidence that LP contaminations are responsible for TLR2 activity. HEK293 cells stably transfected with bovine TLR2 and TLR1 (e.g. clone 1) did not respond to ultra-pure Escherichia coli LPS preparations but acquired responsiveness when stimulated with differently purified commercial LPS. Thus, the described system involving HEK293 cells stably transfected with bovine TLR2 and TLR1 is the first test system described attempting to measure a contamination by LP.
Veterinary Immunology and Immunopathology 10/2011; 145(1-2):66-73. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors are pattern recognition receptors with which hosts recognize pathogen-associated molecular patterns (PAMP). This recognition process is translated rapidly into a meaningful defense reaction. This form of innate host defense is preserved in the animal kingdom: invertebrates heavily depend on it; higher vertebrates also have an adaptive immune system. Both adaptive and innate immune systems are intertwined in that the former also depends on an intact innate recognition and response system. Members of the TLR system cover recognition of parasitic, bacterial or viral germs. Due to the constraints imposed by the necessity to recognize PAMP and to interact with downstream signaling molecules, the TLR system is relatively conserved in evolution. Nevertheless, subtle species differences have been reported for several mammalian TLR members. Examples of this will be given. In all mammalian species investigated, part of the coding sequence is available for the most important TLR members, thus allowing study of expression of these TLR members in various tissues by reverse-transcription polymerase chain reaction in its classical (RT-PCR) and quantitative real time RT-PCR (qRT-PCR) form. In some species, the whole coding sequences of the most important or even all TLR members are known. This allows construction of cDNA and transfection of common host cells, thus permitting functional studies. Extensive investigations were devoted to the study of non-synonymous single nucleotide polymorphisms. In a few cases, expression of a given amino acid in the extracellular (ligand-binding) portion of TLR members could be associated with infectious diseases. This will be discussed below.
Cell and Tissue Research 10/2010; 343(1):107-20. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLR) are highly conserved pattern recognition receptors of the innate immune system. Toll-like receptor 2 (TLR2) recognizes bacterial lipopeptides in a heterodimeric complex with TLR6 or TLR1, thereby discriminating between di- or triacylated lipopeptides, respectively. Previously, we found that HEK293 cells transfected with bovine TLR2 (boTLR2) were able to respond to diacylated lipopeptides but did not recognize triacylated lipopeptides, even after cotransfection with the so far published sequence of boTLR1. In this study we now could show that primary bovine cells were in general able to detect triacylated lipopetides. A closer investigation of the boTLR1 gene locus revealed an additional ATG 195 base pairs upstream from the published start codon. Its transcription would result in an N-terminus with high identity to human and murine TLR1 (huTLR1, muTLR1). Cloning and cotransfection of this longer boTLR1 with boTLR2 now resulted in the recognition of triacylated lipopeptides by HEK293 cells, thereby resembling the ex vivo observation. Analysis of the structure-activity relationship showed that the ester-bound acid chains of these lipopeptides need to consist of at least 12 carbon atoms to activate the bovine heterodimer showing similarity to the recognition by huTLR2/huTLR1. In contrast, HEK293 cell cotransfected with muTLR2 and muTLR1 could already be activated by lipopeptides with shorter fatty acids of only 6 carbon atoms. Thus, our data indicate that the additional N-terminal nucleotides belong to the full length and functionally active boTLR1 (boTLR1-fl) which participates in a species-specific recognition of bacterial lipopeptides.
Veterinary Research 05/2010; 41(3):34. · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but <or=10% of their offspring born in Europe (2nd generation) do so. Recently, we showed that PBMC from 1st generation horses produce more IL-4 than 2nd generation horses. Since helminths and allergens induce Th2 responses, we investigated whether horses domiciled in Iceland are Th2-biased, and whether this is determined by helminth infection. We compared the parasite burden and T cell responses between Icelandic horses living in Iceland or Switzerland. Horses in Iceland have higher faecal egg counts, higher tapeworm-specific IgG(T) levels and higher total serum IgE levels than horses in Switzerland. Nevertheless, horses in Iceland displayed a low proportion of IL-4-producing cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.
Veterinary Immunology and Immunopathology 07/2009; 133(1):40-50. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns on microbes, and ligand recognition results in production of pro-inflammatory cytokines, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. The purpose of this study was to test antibodies specific for human or murine TLR2, 3, 4, 5 and 9 for their use in dogs. Twenty-two antibodies were assessed for recognition of canine monocytes (Mo), granulocytes (Gr) or lymphocytes (Ly) by flow cytometry, and results were compared with isotype-matched controls and other antibodies against the same TLR. Nine monoclonal antibodies detected canine leukocyte subpopulations. Antibodies TL2.1 and TL2.3 (specific for human TLR2), HTA125 (specific for human TLR4), as well as 85B152.5 and 19D759.2 (specific for human TLR5) recognized Mo and Gr but not Ly without permeabilization, and putatively cross-react with canine TLR2, 4 and 5, respectively. Antibodies 40C1285.6 and TLR3.7 (specific for human TLR3) as well as 26C593.2 and 5G5 (specific for human and murine TLR9) recognized Mo, Gr and Ly after their permeabilization and putatively cross-react with canine TLR3 and 9, respectively, inside the cell. None of these nine antibodies recognized paraformaldehyde-treated (4%) canine leukocytes but all except 40C1285.6, TLR3.7 and 5G5 recognized methanol-fixed cells, suggesting that they might be useful also in immunohistochemistry.
Veterinary Immunology and Immunopathology 08/2008; 124(1-2):184-91. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.
Veterinary Immunology and Immunopathology 07/2008; 125(3-4):326-36. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown.
That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD.
Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea.
Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs.
There were significant differences (P< or = .017) in expression of TLR2, 4, and 9 between the 6 sampled locations in healthy control dogs (lymph node > small intestine > or = colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores.
Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.
Journal of Veterinary Internal Medicine 05/2008; 22(3):553-60. · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophages play major roles in the onset of immune responses and inflammation by inducing a variety of cytokines such as TNF and IFN-beta. The pathogen-associated molecular pattern, polyinosinic-polycytidylic acid [poly(I:C)], and LPS were used to study type-I IFN and TNF responses in human macrophages. Additionally, activation of the key signaling pathways, IFN-regulatory factor 3 (IRF3) and NF-kappaB, were studied. We found that TNF production occurred rapidly after LPS stimulation. LPS induced a strong IFN-beta mRNA response within a short time-frame, which subsided at 8 h. The IFN-stimulated genes (ISGs), ISG56 and IFN-inducible protein 10, were strongly induced by LPS. These responses were associated with NF-kappaB and IRF3 activation, as shown by IRF3 dimerization and by nuclear translocation assays. poly(I:C), on the other hand, induced a strong and long-lasting (>12 h) IFN-beta mRNA and protein response, particularly when transfected, whereas only a protracted TNF response was observed when poly(I:C) was transfected. However, these responses were induced in the absence of detectable IRF3 and NF-kappaB signaling. Thus, in human macrophages, poly(I:C) treatment induces a distinct cytokine response when compared with murine macrophages. Additionally, a robust IFN-beta response can be induced in the absence of detectable IRF3 activation.
Journal of Leukocyte Biology 05/2008; 83(5):1249-57. · 4.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of insects of the genus Culicoides. IBH does not occur in Iceland due to the absence of Culicoides. However, Icelandic horses exported to mainland Europe as adults (1st generation) have a > or =50% incidence of developing IBH. In contrast, their progeny (2nd generation) has a <10% incidence of IBH. Here we show that peripheral blood mononuclear cells (PBMC) from Icelandic horses born in mainland Europe and belonging either to the IBH or healthy subgroup produce less interleukin (IL)-4 after polyclonal or allergen-specific stimulation when compared with counterparts from horses born in Iceland. We examined a role of IL-10 and transforming growth factor (TGF)-beta1 in down-regulation of IL-4 in healthy 2nd generation Icelandic horses. Supernatants of PBMC from 2nd generation healthy horses down-regulated the proportion of IL-4-producing cells and IL-4 production in stimulated cultures of PBMC from 1st generation IBH. This inhibition was mimicked by a combination of IL-10 and TGF-beta1 but not by the single cytokines. Cultures of stimulated PBMC of healthy 2nd generation horses produced a low level of IL-4, but IL-4 production was increased by anti-equine IL-10 and anti-human TGF-beta1. This shows for the first time that in horses, IL-10 and TGF-beta1 combined regulate IL-4 production in vitro. It is suggested that in this naturally occurring IgE-mediated allergy, IL-10 and TGF-beta1 have a role in the down-regulation of IL-4-induced allergen-specific Th2 cells, thereby reducing the incidence of IBH.
Veterinary Immunology and Immunopathology 03/2008; 122(1-2):65-75. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induction of interferon-beta (IFN-beta) gene expression is a tightly regulated process, and a plethora of studies identified the signal transduction pathway TANK-binding kinase-1 (TBK-1)/IFN regulatory factor-3 (IRF-3) as essential to the induction of IFN-beta gene expression. Data regarding the role of p38 and JNK are rare, however. We investigated the contribution of these kinases to IFN-beta expression in human macrophages treated with poly(I:C), lipopolysaccharide (LPS), Sendai virus, or vesicular stomatitis virus (VSV). We found that all the stimuli induced IFN-beta mRNA, albeit to a different extent. Whereas LPS and VSV induced the phosphorylation of p38 and JNK, neither poly(I:C) nor Sendai virus led to the detection of phosphospecific signals. When inhibiting p38, a VSV-triggered IFN-beta mRNA response was inhibited, whereas inhibiting JNK suppressed an LPS-triggered response, but only when macrophages were primed with IFN-gamma. Neither poly(I:C)-induced nor Sendai virus-induced IFN-beta mRNA expression was affected when p38 and JNK were inhibited. Collectively, the data show that the contribution of p38 and JNK to the expression of IFN-beta occurs in a stimulation-specific manner in human macrophages.
Journal of Interferon & Cytokine Research 10/2007; 27(9):751-5. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.
Veterinary Immunology and Immunopathology 08/2007; 118(1-2):92-104. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.
The Journal of Immunology 08/2007; 179(2):1166-77. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Knowledge of the role of origin-related, environmental, sex, and age factors on host defence mechanisms is important
to understand variation in parasite intensity. Because alternative components of parasite defence may be differently
sensitive to various factors, they may not necessarily covary. Many components should therefore be considered to
tackle the evolution of host–parasite interactions. In a population of barn owls (
), we investigated the role
of origin-related, environmental (i.e. year, season, nest of rearing, and body condition), sex, and age factors on 12
traits linked to immune responses [humoral immune responses towards sheep red blood cells (SRBC), human serum
albumin (HSA) and toxoid toxin TT, T-cell mediated immune response towards the mitogen phytohemagglutinin
(PHA)], susceptibility to ectoparasites (number and fecundity of
, number of
disease symptoms (size of the bursa of Fabricius and spleen, proportion of proteins that are immunoglobulins, haematocrit
and blood concentration in leucocytes). Cross-fostering experiments allowed us to detect a heritable component
of variation in only four out of nine immune and parasitic parameters (i.e. SRBC- and HSA-responses,
haematocrit, and number of
). However, because nestlings were not always cross-fostered just after
hatching, the finding that 44% of the immune and parasitic parameters were heritable is probably an overestimation.
These experiments also showed that five out of these nine parameters were sensitive to the nest environment
(i.e. SRBC- and PHA-responses, number of
, haematocrit and blood concentration in leucocytes).
Female nestlings were more infested by the blood-sucking fly
than their male nestmates, and their
blood was less concentrated in leucocytes. The effect of year, season, age (i.e. reflecting the degree of maturation of
the immune system), brood size, position in the within-brood age hierarchy, and body mass strongly differed between
the 12 parameters. Different components of host defence mechanisms are therefore not equally heritable and
sensitive to environmental, sex, and age factors, potentially explaining why most of these components did not covary.
Biological Journal of the Linnean Society 04/2007; 90:703-718. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Equine insect bite hypersensitivity (IBH) is an immediate-type hypersensitivity reaction provoked by insect-derived allergens. Icelandic horses living in Iceland do not have IBH due to absence of relevant insects, but acquire it at high frequency after being imported to mainland Europe. In contrast, their offspring born in mainland Europe has reduced IBH incidence. T helper 1 (Th1) and Th2 cells and cytokines were determined in Icelandic horses born in Iceland and on the continent and which either have IBH or are healthy.
Peripheral blood mononuclear cells (PBMC) from these horses were stimulated for 18 h during summer and winter with polyclonal T cell stimuli, IBH allergen(s) or irrelevant allergen(s). Cells were analysed by flow cytometry for interferon-gamma (IFN-gamma) and interleukin-4 (IL-4); RNA was analysed for IFN-gamma, IL-4, IL-5 and IL-13 mRNA.
During summer, but not during winter, IBH PBMC stimulated polyclonally showed reduced IFN-gamma mRNA and IFN-gamma-producing cells when compared with those of healthy horses, regardless of origin. PBMC stimulated polyclonally or with IBH allergen showed increased IL-4 mRNA levels and higher numbers of IL-4-producing cells when born in Iceland or showing IBH symptoms. IL-5 and IL-13 mRNA were modulated neither by disease nor by origin. Abrogation of IL-4 production in healthy horses born in mainland Europe may be due, at least in part, to IL-10. There was an increased level of IL-10 in supernatants from PBMC of healthy horses born in mainland Europe and stimulated polyclonally or with IBH allergen.
Modulation of IBH incidence is governed by altered Th1/Th2 ratio, which might be influenced by IL-10.
International Archives of Allergy and Immunology 02/2007; 144(4):325-37. · 2.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human whole blood IL-1 test exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material. Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after incubation. In order to understand any differences between the pyrogenic activity in this test and the existing live rabbit test (species differences versus aberrant response of the particular blood sample), the rabbit whole blood test was developed. This approach could also help to avoid the use of putatively infectious human blood for pyrogen testing in vitro.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.
Veterinary Immunology and Immunopathology 12/2006; 114(1-2):1-14. · 1.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Osteoarthritis due to cranial cruciate ligament (CCL) rupture or hip dysplasia is one of the most important causes of chronic lameness in dogs. This study aimed at comparing nitric oxide (NO) production by the CCL with that of the femoral head ligament (FHL) and the medial collateral ligament (MCL), and investigating the pathway of NO production and the concomitant metalloproteinase (MMP) activity in the presence or absence of an inflammatory stimulus. Ligaments of normal dogs were subjected to different stimuli, and NO and MMP activity from explant culture supernatants were compared. The results showed that in explant cultures of the canine CCL more NO was produced than in those of the other two ligaments. A higher level of NO was produced when CCLs were exposed to the inducible nitric oxide synthase (iNOS)-inducing cocktail TNF/IL-1/LPS, and NO synthesis could be inhibited by both l-NMMA, a general nitric oxide synthase (NOS) inhibitor and l-NIL, a specific iNOS inhibitor. However, a correlation between NO synthesis and iNOS expression levels as determined by immunohistochemistry was not observed. In contrast to CCL, no evidence for iNOS-dependent NO synthesis was observed for MCL and FHL. The CCL produced less MMP than MCL and FHL, and no correlation between MMP and NO could be demonstrated. MMP activity in the CCL increased significantly after 48 h of incubation with the inflammatory stimulus. The results suggest that in canine osteoarthritis NO synthesized by canine CCL plays a more important role in the pathogenesis of osteoarthritis of the stifle than that synthesized by FHL and MCL.
The Veterinary Journal 12/2006; 172(3):466-72. · 2.17 Impact Factor