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ABSTRACT: Recent results call for the reexamination of the phenotype of wound macrophages and their role in tissue repair. These results include the characterization of distinct circulating monocyte populations with temporally restricted capacities to migrate into wounds and the observation that the phenotype of macrophages isolated from murine wounds partially reflects those of their precursor monocytes, changes with time, and does not conform to current macrophage classifications. Moreover, findings in genetically modified mice lacking macrophages have confirmed that these cells are essential to normal wound healing because their depletion results in retarded and abnormal repair. This mini-review focuses on current knowledge of the phenotype of wound macrophages, their origin and fate, and the specific macrophage functions that underlie their reparative role in injured tissues, including the regulation of the cellular infiltration of the wound and the production of transforming growth factor-β and vascular endothelial growth factor.
American Journal Of Pathology 01/2011; 178(1):19-25. · 4.89 Impact Factor
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ABSTRACT: The phenotype of wound macrophages has not been studied by direct examination of these cells, yet macrophages recruited to sites of injury are described as alternatively activated macrophages, requiring IL-4 or IL-13 for phenotypic expression. This study characterized wound macrophage phenotype in the PVA sponge wound model in mice. Eighty-five percent of wound macrophages isolated 1 day after injury expressed Gr-1, but only 20% of those isolated at 7 days expressed this antigen. Macrophages from 1-, 3-, and 7-day wounds expressed markers of alternative activation,including mannose receptor, dectin-1, arginase 1,and Ym1, but did not contain iNOS. Day 1 wound macrophages produced more TNF-alpha, more IL-6, and less TGF-beta than Day 7 wound macrophages. Wound macrophages did not produce IL-10. The cytokines considered necessary for alternative activation of macrophages,IL-4 and IL-13, were not detected in the wound environment and were not produced by wound cells.Wound macrophages did not contain PStat6. Wound fluids inhibited IL-13-dependent phosphorylation of Stat6 and contained IL-13Ralpha2, a soluble decoy receptor for IL-13. The phenotype of wound macrophages was not altered in mice lacking IL-4Ralpha, which is required for Stat6-dependent signaling of IL-4 and IL-13.Wound macrophages exhibit a complex phenotype,which includes traits associated with alternative and classical activation and changes as the wound matures.The wound macrophage phenotype does not require IL-4 or IL-13.
Journal of leukocyte biology 01/2010; 87(1):59-67. · 4.99 Impact Factor
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ABSTRACT: In this study, we investigated the role of interleukin (IL)-1 signaling in wound healing. IL-1 receptor type I (IL-1R) knockout (KO) mice showed reduced fibrosis in both cutaneous and deep tissue wounds, which was accompanied by a reduction in inflammatory cellular infiltration in cutaneous but not in deep tissue wounds. There were no differences in either total collagenolytic activity or in the expression of selected matrix metalloproteinases or tissue inhibitors of metalloproteinases between the wound fluids from wild-type or IL-1R KO mice. However, wound fluids from IL-1R KO mice contained lower levels of IL-6 compared with wild-type controls. In addition, the infusion of IL-6 into wounds in IL-1R KO mice did not increase fibrosis. Skin wounds in IL-1R KO animals had lower levels of collagen and improved restoration of normal skin architecture compared with skin wounds in wild-type mice. However, neither the tensile strength of incisional skin wounds nor the rate of closure of excisional wounds differed between IL-1R KO and wild-type animals. The reduced fibrotic response in wounds from IL-1R KO mice could be reproduced by the administration of an IL-1R antagonist. These findings suggest that pharmacological interference with IL-1 signaling could have therapeutic value in the prevention of hypertrophic scarring and in the treatment of fibrotic diseases.
American Journal Of Pathology 05/2009; 174(6):2129-36. · 4.89 Impact Factor
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ABSTRACT: Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE(2), which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE(2) on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE(2) strongly suppresses LPS-induced IFN-beta production at the mRNA and protein levels. Poly (I:C)-induced IFN-beta and LPS-induced CCL5 production were also suppressed by PGE(2). The inhibitory effect of PGE(2) on LPS-induced IFN-beta expression is mediated through PGE(2) receptor subtypes EP(2) and EP(4), and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3beta. In contrast, PGE(2) inhibits LPS-induced TNF-alpha production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-beta concentration in mice. Collectively, data demonstrate that PGE(2) is a negative regulator for IFN-beta production in activated macrophages and during endotoxemia.
The Journal of Immunology 03/2008; 180(4):2125-31. · 5.79 Impact Factor
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ABSTRACT: The anti-granulocyte receptor-1 (Gr-1) mAb, RB6-8C5, has been used extensively to deplete neutrophils in mice and to investigate the role of these cells in host defense. RB6-8C5 binds to Ly6G, which is present on neutrophils, and to Ly6C, which is expressed on neutrophils, dendritic cells, and subpopulations of lymphocytes and monocytes. It is thus likely that in vivo administration of RB6-8C5 may deplete not only neutrophils but also other Gr-l+ (Ly6C+) cells. This study describes the use of an Ly6G-specific mAb, 1A8, as an alternative means to deplete neutrophils. In vivo administration of RB6-8C5 reduced blood neutrophils and Gr-1+ monocytes, whereas administration of 1A8 reduced blood neutrophils but not Gr-1+ monocytes. Plasma TNF-alpha in endotoxemia was increased 20-fold by RB6-8C5 pretreatment and fourfold by 1A8 pretreatment. In a wound model, pretreatment with either antibody decreased wound neutrophils and macrophages. TNF-alpha staining in brefeldin-treated wound leukocytes was increased by pretreatment with RB6-8C5, but not 1A8. Neutrophil depletion with 1A8 offers advantages over the use of RB6-8C5, as it preserves non-neutrophil Gr-1+ cells depleted by the anti-Gr-1 antibody. The loss of non-neutrophil Gr-1+ populations in RB6-8C5-treated animals is associated with increased TNF-alpha responses, suggesting these cells may function to suppress TNF-alpha production.
Journal of Leukocyte Biology 02/2008; 83(1):64-70. · 4.99 Impact Factor
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ABSTRACT: Extravasation of leukocytes from peripheral blood is required for an effective inflammatory response at sites of tissue infection. Integrins help mediate extravasation and navigate the leukocyte to the infectious source. A novel role for integrins in regulating the effector response to a cell wall component of fungal pathogens is the subject of the current study. Although phagocytosis is useful for clearance of unicellular fungi, the immune response against large, noningestible hyphae is not well-understood. Fungal beta-glucan, a pathogen-associated molecular pattern, activates production of superoxide anion in leukocytes without the need for phagocytosis. To model polymorphonuclear leukocyte (PMN) recognition of fungi under conditions in which phagocytosis cannot occur, beta-glucan was covalently immobilized onto tissue culture plastic. Plasma membrane-associated respiratory burst was measured by reduction of ferricytochrome C. Results show that the human PMN oxidative burst response to immobilized beta-glucan is suppressed by addition of beta(1) integrin ligands to the beta-glucan matrix. Suppression was dose dependent and steric hindrance was ruled out. beta(1) integrin ligands did not affect respiratory burst to ingestible beta-glucan-containing particles, phorbol esters or live yeast hyphae. Furthermore, in the absence of matrix, Ab activation of VLA3 or VLA5, but not other beta(1) integrins, also prevented beta-glucan-induced respiratory burst. beta(1)-induced suppression was blocked and burst response restored by treating neutrophils with either the cell-binding fragment of soluble human Fn, cyclic RGD peptide, or Ab specific to VLA3 or VLA5. Together these findings extend the functional role of beta(1) integrins to include modulating PMN respiratory burst to a pathogen-associated molecular pattern.
The Journal of Immunology 07/2007; 178(11):7276-82. · 5.79 Impact Factor
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ABSTRACT: Candida albicans is a common cause of nosocomial infections whose virulence depends on the reversible switch from blastoconidia to hyphal forms. Neutrophils (or polymorphonuclear leukocytes (PMNs)) readily clear blastoconidia by phagocytosis, but filaments are too long to be ingested. Mechanisms regulating immune recognition and response to filamentous fungal pathogens are not well understood, although known risk factors for developing life-threatening infections are neutropenia or defects in the NADPH oxidase system. We show human PMNs generate a respiratory burst response to unopsonized hyphae. Ab specific for beta-glucan, a major component of yeast cell walls, blocks this response, establishing beta-glucan as a key molecular pattern recognized by PMNs in response to C. albicans. This study also elucidates recognition and signaling mechanisms used by PMNs in response to beta-glucan under conditions where phagocytosis cannot occur. Human PMNs adhered to immobilized beta-glucan and released an efficient plasma membrane respiratory burst. Ab blockade of the integrin complement receptor 3 (CD11b/CD18) significantly inhibited both of these functions. Furthermore, we show a role for p38 MAPK and actin but not protein kinase C zeta in generating the respiratory burst to beta-glucan. Taken together, results show that beta-glucan in C. albicans hyphae is accessible to PMNs and sufficient to support an innate immune response.
The Journal of Immunology 01/2007; 177(12):8667-75. · 5.79 Impact Factor
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Jorge E Albina
Nutrition in Clinical Practice 01/2007; 21(6):623-5. · 1.59 Impact Factor
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ABSTRACT: The beta-glucans are long-chain polymers of glucose in beta-(1,3)(1,6) linkages, which comprise the fungal cell wall and stimulate cells of the innate immune system. Previous in vitro studies have shown the ability of beta-glucan to increase the chemotactic capacity of human neutrophils. The current study examined an in vivo correlate of that observation by testing the hypothesis that systemic beta-glucan treatment would result in enhanced migration of neutrophils into a site of inflammation and improve antimicrobial function. A model of acute inflammation was used in which polyvinyl alcohol sponges were implanted subcutaneously into the dorsum of rats. Animals treated with beta-glucan showed a 66 +/- 6% and 186 +/- 42% increase in wound cell number recovered 6 and 18 h postwounding, respectively. Increased migration did not correlate with increased chemoattractant content of wound fluid, alterations in neutrophil-induced loss of endothelial barrier function, or changes in neutrophil adhesion to endothelial cells. Systemic administration of SB203580 abrogated the enhanced migration by beta-glucan without altering normal cellular entry into the wound. Studies also showed a priming effect for chemotaxis and respiratory burst in circulating neutrophils isolated from beta-glucan-treated animals. Heightened neutrophil function took place without cytokine elicitation. Furthermore, beta-glucan treatment resulted in a 169 +/- 28% increase in neutrophil number and a 60 +/- 9% decrease in bacterial load in the bronchoalveolar lavage fluid of Escherichia coli pneumonic animals. Taken together, these findings demonstrate that beta-glucan directly affects the chemotactic capacity of circulating neutrophils through a p38 mitogen-activated protein kinase-dependent mechanism and potentiates antimicrobial host defense.
Journal of Leukocyte Biology 05/2006; 79(4):667-75. · 4.99 Impact Factor
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ABSTRACT: Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.
Molecular and Cellular Biology 07/2005; 25(11):4716-26. · 5.53 Impact Factor
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ABSTRACT: Neutropenia has been shown to markedly increase plasma TNF-alpha concentration after LPS injection and to enhance LPS-induced mortality. Experiments reported here demonstrate that the 15-fold higher plasma TNF-alpha concentration elicited by LPS in neutropenic vs. nonneutropenic unanesthetized mice correlated with increased hepatic and splenic, but not pulmonary, TNF-alpha mRNA. Core 2 beta-1,6-N-acetylglucosaminyltransferase-null and CD18-deficient mice also exhibited exaggerated plasma TNF-alpha responses to LPS injection. Findings suggest that extravasated neutrophils inhibit systemic TNF-alpha production and that they do so through organ-selective mechanisms involving CD18 integrin and selectin binding.
AJP Regulatory Integrative and Comparative Physiology 03/2005; 288(2):R409-12. · 3.34 Impact Factor
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ABSTRACT: Arginase activity is expressed by macrophages in healing wounds and other sites of inflammation and has been shown to modulate the synthesis of nitric oxide, polyamines, and collagen. The role of CCAAT/enhancer-binding protein beta (C/EBPbeta) in the regulation of macrophage arginase by different agonists was investigated using C/EBPbeta-/- and +/+ macrophage cell lines. 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP, 0.5 mM), recombinant murine interleukin 4 (rmIL-4, 20 U/mL), Escherichia coli lipopolysaccharide (100 ng/mL), and hypoxia (1% O2) induced arginase activity in C/EBPbeta+/+ macrophages, where enzyme activity correlated with arginase I protein. Only rmIL-4 increased arginase activity in C/EBPbeta-/- cells. Arginase II protein was expressed constitutively in wild-type and C/EBPbeta-/- cell lines and was unaltered by 8-Br-cAMP or rmIL-4. rmIL-4-stimulated immortalized C/EBPbeta-/- macrophages demonstrated higher nuclear signal transducer and activator of transcription-6 (STAT6) and phospho-STAT6 content than their +/+ counterparts. Validating the biological relevance of findings with the cell lines, additional experiments examined wound fluids and peritoneal macrophages from C/EBPbeta-/- mice and demonstrated that both contained less arginase activity than those from wild-type controls. Wounds in C/EBPbeta-/- animals showed signs of delayed maturation, as manifested by the persistence of neutrophils in the inflammatory infiltrate. Peritoneal macrophages from C/EBPbeta+/+ animals responded to 8-Br-cAMP and rmIL-4 with increased arginase activity, whereas those from C/EBPbeta-/- mice did not respond to cAMP. Results demonstrate a key mechanistic role for C/EBPbeta in the modulation of macrophage arginase I expression in vivo and in vitro.
Shock 03/2005; 23(2):168-72. · 2.85 Impact Factor
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ABSTRACT: The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-alpha, 168% more IL-6, and 61% less TGF-beta1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-alpha staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-alpha and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-alpha and IL-6 release from LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-alpha release from LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-alpha and superoxide release from murine peritoneal macrophages. The TNF-alpha inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.
The Journal of Immunology 02/2005; 174(4):2265-72. · 5.79 Impact Factor
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ABSTRACT: Dendritic cells (DCs) are professional antigen-presenting cells that act as sentinels in the cell-mediated response against invading pathogens associated with septic challenge. The purpose of the present study was to determine whether there is a loss of dendritic cells and/or changes in function of these cells in septic mice. Here we report that the number of DCs, in both spleen and peritoneum, decreased over 24 h postsepsis [cecal ligation and puncture (CLP)] when compared with sham. The most dramatic change was seen in the peritoneal cavity. This decrease appeared to be caused mainly by the depletion of immature DCs rather than mature DCs. This change was LPS independent and minimally affected by FasL; however, overexpression of human Bcl-2 gene provides protection of the septic peritoneal DCs. Moreover, although the level of IL-12 release decreased significantly in splenic DCs obtained from CLP mice, IL-12 secretion was markedly elevated by peritoneal DCs as well as in both plasma and peritoneal fluid at 24 h post-CLP. In peritoneal cells, the expression of CD40, CD80, and CD86 was unchanged, but their respective ligands CD40L, CD28, and CD152 all increased in mice 24 h after CLP, although no such change was observed in splenocytes. Regardless of the presence or absence of antigen, peritoneal DCs from CLP mice showed higher capacity to stimulate T-cell proliferation than those cells from the sham control. However, splenic DCs from CLP mice only showed augmented capacity to induce antigen-dependent stimulation of T-cell proliferation. Together, these data indicate that sepsis produces divergent functional changes in splenic and peritoneal DC populations.
Shock 09/2004; 22(2):137-44. · 2.85 Impact Factor
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ABSTRACT: Polymorphonuclear leukocytes (PMNs) must traverse endogenous chemotactic gradients (interleukin 8 [IL-8]) before reaching target chemoattractants (fMLP [N-formylmethionine-leucine-phenylalanine], C5a) produced at a site of bacterial infection. Complement receptor 3 (CR3; CD11b/CD18) contains 2 distinct binding sites, one that mediates adhesion and a lectin-like domain (LLD) that binds polysaccharides of microbial origin. This laboratory previously reported an increase in the chemotactic capacity of PMNs toward fMLP upon ligation of the CR3 LLD with beta-glucan, a CR3 agonist. Current studies sought to determine the effect of beta-glucan on PMN navigation toward other chemoattractants alone and in a competing chemotactic environment.
Migration was assessed by serum agarose overlay with the use of chambered slides containing or not, beta-glucan. Migration of human PMNs at 37 degrees C for 2 hours was evaluated toward C5a or IL-8 alone and in competing gradients. Selected groups were treated with anti-CR3-blocking antibodies. The number of chemotactic cells was quantified by microscopy.
beta-glucan significantly enhanced chemotaxis toward C5a and suppressed that toward IL-8 in a CR3-dependent fashion. In the competing chemotactic gradient assays (C5a vs IL-8), beta-glucan further enhanced migration toward C5a while not affecting that toward IL-8.
beta-glucan selectively upregulates PMN chemotaxis toward C5a while suppressing chemotaxis toward IL-8.
Surgery 09/2004; 136(2):384-9. · 3.10 Impact Factor
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ABSTRACT: The adhesion of neutrophils to endothelial cells is a central event leading to diapedesis and involves the binding of the I-domain of beta(2) integrins (CD11/CD18) to endothelial ICAMs. In addition to the I-domain, the beta(2) integrin complement receptor 3 (CR3) (CD11b/CD18) contains a lectin-like domain (LLD) that can alter leukocyte functions such as chemotaxis and cytotoxicity. The present study demonstrates that, in contrast to the CR3 I-domain, Ab blockade of the CR3 LLD has no role in mediating neutrophil-induced loss of endothelial barrier function. However, activation of CR3 with the LLD agonist beta-glucan protects the barrier function of endothelial cells in the presence of activated neutrophils and reduces transendothelial migration without affecting adhesion of the neutrophils to the endothelium. The LLD site-specific mAb VIM12 obviates beta-glucan protection while activation of the LLD by VIM12 cross-linking mimics the beta-glucan response by both preserving endothelial barrier function and reducing neutrophil transendothelial migration. beta-glucan has no direct effect on endothelial cell function in the absence of activated neutrophils. These findings demonstrate that signaling through the CR3 LLD prevents neutrophil-induced loss of endothelial barrier function and reduces diapedesis. This suggests that the LLD may be a suitable target for oligosaccharide-based anti-inflammatory therapeutics.
The Journal of Immunology 08/2004; 173(2):1284-91. · 5.79 Impact Factor
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Methods in Enzymology 02/2004; 381:527-38. · 2.04 Impact Factor
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Methods in molecular medicine 02/2003; 78:369-76.
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ABSTRACT: Work from Tom Hunt's laboratory first identified wound hypoxia as a potential regulator of the biology of cells participating in tissue repair. Current understanding of the role of oxygen in the regulation of gene expression begins to provide a mechanistic basis for the prediction that oxygen could be a fundamental regulator of wound healing made by the Hunt laboratory. The present article describes the experience of the authors' laboratory in defining the expression of two oxygen-regulated genes, those for the inducible form of nitric oxide synthase and for arginase I in experimental wounds. Observations made regarding these two genes are discussed in the context of the overall regulatory role of oxygen as a phenotypic modulator of inflammatory cells.
Wound Repair and Regeneration 11(6):445-51. · 2.91 Impact Factor
