CD19, a Response Regulator of B Lymphocytes, Regulates Wound Healing through Hyaluronan-Induced TLR4 Signaling
2009; Elsevier BV; Volume: 175; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2009.080355
ISSN1525-2191
AutoresYohei Iwata, Ayumi Yoshizaki, Kazuhiro Komura, Kazuhiro Shimizu, Fumihide Ogawa, Toshihide Hara, Eiji Muroi, SangJae Bae, Motoi Takenaka, Toru Yukami, Minoru Hasegawa, Manabu Fujimoto, Yasushi Tomita, Thomas F. Tedder, Shinichi Sato,
Tópico(s)Immune Response and Inflammation
ResumoImmune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-β. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-β through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing. Immune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-β. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-β through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing. Healing of cutaneous wounds is a complex biological event that results from the interplay of a large number of resident and infiltrating cell types, including leukocytes.1Gillitzer R Goebeler M Chemokines in cutaneous wound healing.J Leukoc Biol. 2001; 69: 513-521Crossref PubMed Google Scholar Accumulating evidence has revealed a critical role of leukocytes in wound healing. Infiltrating neutrophils form a first line of defense against local infections and are also a source of pro-inflammatory cytokines to activate fibroblasts and keratinocytes.2Hubner G Brauchle M Smola H Madlener M Fassler R Werner S Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.Cytokine. 1996; 8: 548-556Crossref PubMed Scopus (396) Google Scholar Macrophages also regulate wound healing by antimicrobial function, wound debridement, and production of various growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, basic fibroblast growth factor (bFGF), heparin binding epidermal growth factor, and TGF-α.3Witte MB Barbul A General principles of wound healing.Surg Clin North Am. 1997; 77: 509-528Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 4Martin P Wound healing–aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3778) Google Scholar, 5Singer AJ Clark RA Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4720) Google Scholar, 6Park JE Barbul A Understanding the role of immune regulation in wound healing.Am J Surg. 2004; 187: 11S-16SAbstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar These factors stimulate the synthesis of extracellular matrix by local fibroblasts, generate new blood vessels, promote the granulation tissue formation, and enhance re-epithelialization.4Martin P Wound healing–aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3778) Google Scholar, 5Singer AJ Clark RA Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4720) Google Scholar Furthermore, a series of experimental studies have indicated a significant role for T lymphocytes in wound healing as growth factor-producing cells as well as immunological effector cells.1Gillitzer R Goebeler M Chemokines in cutaneous wound healing.J Leukoc Biol. 2001; 69: 513-521Crossref PubMed Google Scholar, 7Blotnick S Peoples GE Freeman MR Eberlein TJ Klagsbrun M T lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by CD4+ and CD8+ T cells.Proc Natl Acad Sci USA. 1994; 91: 2890-2894Crossref PubMed Scopus (279) Google Scholar, 8Schaffer M Barbul A Lymphocyte function in wound healing and following injury.Br J Surg. 1998; 85: 444-460Crossref PubMed Scopus (171) Google Scholar, 9Boyce DE Jones WD Ruge F Harding KG Moore K The role of lymphocytes in human dermal wound healing.Br J Dermatol. 2000; 143: 59-65Crossref PubMed Scopus (80) Google Scholar Thus, immune cells have an integral function in wound healing beyond their role in inflammation and host defense, mainly through the secretion of signaling molecules, such as cytokines and growth factors.6Park JE Barbul A Understanding the role of immune regulation in wound healing.Am J Surg. 2004; 187: 11S-16SAbstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar However, little is known regarding a role of B cells in wound healing. Previous studies have revealed that B cells are present within wound tissue9Boyce DE Jones WD Ruge F Harding KG Moore K The role of lymphocytes in human dermal wound healing.Br J Dermatol. 2000; 143: 59-65Crossref PubMed Scopus (80) Google Scholar, 10Cowin AJ Brosnan MP Holmes TM Ferguson MW Endogenous inflammatory response to dermal wound healing in the fetal and adult mouse.Dev Dyn. 1998; 212: 385-393Crossref PubMed Scopus (163) Google Scholar, 11Richards AM Floyd DC Terenghi G McGrouther DA Cellular changes in denervated tissue during wound healing in a rat model.Br J Dermatol. 1999; 140: 1093-1099Crossref PubMed Scopus (52) Google Scholar and that B cell count is rapidly increased in the first 4 days after wounding, and thereafter reaches a plateau before falling as the wounds heal.11Richards AM Floyd DC Terenghi G McGrouther DA Cellular changes in denervated tissue during wound healing in a rat model.Br J Dermatol. 1999; 140: 1093-1099Crossref PubMed Scopus (52) Google Scholar Furthermore, recent assessment of the role of B cells in the immune system has indicated that B cells are more than just the precursors of antibody (Ab)-secreting cells.12Lipsky PE Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity.Nat Immunol. 2001; 2: 764-766Crossref PubMed Scopus (484) Google Scholar B cells have essential functions in regulating immune responses, including the production of various cytokines and growth factors, antigen presentation, regulation of T cell activation and differentiation, and regulation of lymphoid organization.12Lipsky PE Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity.Nat Immunol. 2001; 2: 764-766Crossref PubMed Scopus (484) Google Scholar Therefore, the increased numbers of B cells within wound tissue may reflect a role for these cells that has hitherto been unrecognized. Both innate and adaptive immune responses contribute to host defense cooperatively. B cells play a principal role in adaptive immune response through B cell antigen receptor (BCR). BCR-induced signals are further modified by other cell surface molecules including CD19. CD19, a major positive response regulator, is a critical B cell-specific signal transduction molecule of the immunoglobulin superfamily expressed by early pre-B cells from the time of heavy chain rearrangement until plasma cell differentiation.13Sato S Hasegawa M Fujimoto M Tedder TF Takehara K Quantitative genetic variation in CD19 expression correlates with autoimmunity.J Immunol. 2000; 165: 6635-6643Crossref PubMed Scopus (279) Google Scholar B cells also primarily participate in innate immunity; indeed, B cells express toll-like receptors (TLRs) and respond to exogenous innate stimuli such as lipopolysaccharide (LPS), a major component of Gram-negative bacteria. CD19 also regulates LPS signaling: B cells from CD19-deficient (CD19−/−) mice are hyporesponsive to most transmembrane signals, including BCR ligation and LPS, while B cells from CD19-transgenic (CD19Tg) mice that overexpress CD19 by ∼threefold are hyperresponsive to these signals.14Engel P Zhou LJ Ord DC Sato S Koller B Tedder TF Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 15Sato S Steeber DA Jansen PJ Tedder TF CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19.J Immunol. 1997; 158: 4662-4669Crossref PubMed Google Scholar Thus, CD19 regulates both adaptive and innate immune responses. In the current study, to clarify the roles of B cells in wound healing, we investigated the wound-healing model in CD19−/− and CD19Tg mice. The results of this study indicate that CD19 controls cytokine and growth factor production by B cells mainly through TLR4 signaling, which was activated by an endogenous TLR4 ligand hyaluronan (HA) increased in the wounded skin, and thereby CD19 regulates the skin wound-healing process. CD19−/− (C57BL/6 × 129) and CD19Tg (C57BL/6 × B6/SJL) mice were generated as described.14Engel P Zhou LJ Ord DC Sato S Koller B Tedder TF Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 16Zhou LJ Smith HM Waldschmidt TJ Schwarting R Daley J Tedder TF Tissue-specific expression of the human CD19 gene in transgenic mice inhibits antigen-independent B-lymphocyte development.Mol Cell Biol. 1994; 14: 3884-3894Crossref PubMed Scopus (123) Google Scholar All mice were healthy, fertile, and did not display any evidence of infection or disease. All mice were backcrossed between 5 to 10 generations onto the C57BL/6 background. Mice were 7 to 12 weeks old for all experiments and age-matched wild-type littermates or C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were used as controls. All mice were housed in a specific pathogen-free barrier facility and screened regularly for pathogens. All studies and procedures were approved by the Committee on Animal Experimentation of Nagasaki University Graduate School of Biomedical Sciences. Mice were anesthetized with diethyl ether and their backs were shaved and wiped with 70% alcohol. Four full-thickness excisional wounds per mouse were made using a disposable sterile 6-mm biopsy punch (Maruho, Osaka, Japan), as described elsewhere.17Subramaniam M Saffaripour S Van De Water L Frenette PS Mayadas TN Hynes RO Wagner DD Role of endothelial selectins in wound repair.Am J Pathol. 1997; 150: 1701-1709PubMed Google Scholar After wounds were covered with an occlusive dressing (Tegaderm, 3M Canada, London, ON), mice were caged individually. At 3 and 7 days after wounding, mice were anesthetized, and areas of open wounds were measured by tracing the wound openings onto a transparency. Any signs suggestive for local infection were not detected in the wounded skin. All four wounds were analyzed for macroscopic analysis of wound closure; however, the wound that was extremely distorted and was difficult to evaluate the correct size was excluded. For macroscopic analysis of wound closure, 50 wounds from 15 mice were used in wild-type group, and 42 wounds from 14 mice were used in CD19−/− and CD19Tg group. After the mice were sacrificed, wounds were harvested with a 2-mm rim of unwounded skin tissue. The wounds were fixed in 3.5% paraformaldehyde and was then paraffin embedded. Six-μm paraffin sections were stained with H&E. The epithelial gap, which represents distance between the leading edge of migrating keratinocytes, was measured under a light microscope. We identified the area that consists of newly formed capillaries and the collection of fibroblasts and macrophages as granulation tissue. Wound sections were visualized in the color monitor (PVM-14M4J, Olympus, Tokyo, Japan) using the CCD camera (CS-900, Olympus). Then, the area of granulation tissue was gated and measured by the video micrometer (VM-60, Olympus). The number of neutrophils and mast cells was counted in the entire section outside the blood vessels by H&E staining and toluidine blue staining. All of the sections were examined independently by two investigators in a blinded fashion. Paraffin-embedded tissues were cut into 6 μm sections, deparaffinized in xylene, and rehydrated in PBS. Deparaffinized sections were treated with endogenous peroxidase blocking reagent (DAKO Cytomation A/S, Copenhagen, Denmark) and proteinase K (DAKO Cytomation A/S) for 6 minutes at room temperature. Sections were then incubated with rat monoclonal Ab (mAb) specific for macrophages (F4/80; American Type Culture Collection, Rockville, MD), CD3 (Dainippon Pharmaceutical Company, Osaka, Japan), and B220 (BD PharMingen, San Diego, CA). Rat IgG (Southern Biotechnology Associates Inc., Birmingham, AL) was used as a control for nonspecific staining. Sections were sequentially incubated with a biotinylated rabbit anti-rat IgG secondary Ab (Vectastain ABC method, Vector Laboratories, Burlingame, CA), then horseradish peroxidase-conjugated avidin-biotin complexes. Sections were washed three times with PBS between incubations. Sections were developed with 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide, and then counterstained with methyl green. Stained cells were counted in nine high-power fields (0.07 mm2, magnification, ×400) in the wound bed per section. Among the nine fields, six fields were selected from both edges of the wound bed, and the remaining three fields were chosen from the middle of the wound bed. Each section was examined independently by two investigators in a blinded fashion. For HA staining, the deparaffinized sections were incubated with 1.5% hydrogen peroxide for 10 minutes to block tissue peroxidase activity. Thereafter, sections were incubated overnight at 4°C with 5 μg/ml of biotinylated HA binding protein (Sigma-Aldrich Co., St. Louis, MO), followed by incubation (30 minutes, 37°C) with streptavidin-horseradish peroxidase (BD PharMingen). Sections were developed and counterstained as described above. For double staining of B cells, the sections were incubated overnight at 4°C with rat anti-mouse B220 Ab (R&D systems, Minneapolis, MN), then sequentially incubated with a biotinylated rabbit anti-rat IgG secondary Ab and horseradish peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC kit, Vector Laboratories). Sections were developed with 3,3′-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Thereafter, sections were incubated overnight at 4°C with goat anti-mouse interleukin (IL)110 Ab (Santa Cruz Biotechnology, Santa Cruz, CA), then sequentially incubated with an alkaline phosphatase-conjugated rabbit anti-goat IgG secondary Ab (Vectastain Elite ABC-AP kit, Vector Laboratories). Sections were developed with Vector Blue. Total RNAs were extracted from wounded skin samples using Qiagen RNeasy spin columns (QIAGEN, Crawley, UK) and subsequently were reverse transcribed to cDNA using Ready-To-Go RT-PCR Beads (GE Health care, Buckinghamshire, UK). Expression of bFGF, acidic FGF (aFGF), IL-6, IL-10, TGF-β1, PDGF, interferon (IFN)-γ, tumor necrosis factor (TNF)- α was quantified by real-time PCR using sequence-specific primers and probes designed by predeveloped TaqMan assay reagents (40 cycles of denaturing at 92°C for 15 seconds and annealing at 60°C for 60 seconds) and an ABI Prism 7300 Sequence Detector (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate was used to normalize mRNA. Relative expression of real-time PCR products was determined by using the ΔΔCt method18Meijerink J Mandigers C van de Locht L Tonnissen E Goodsaid F Raemaekers J A novel method to compensate for different amplification efficiencies between patient DNA samples in quantitative real-time PCR.J Mol Diagn. 2001; 3: 55-61Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar to compare target gene and housekeeping gene mRNA expression. One of the control samples was chosen as a calibrator sample. Splenic B cells were purified (>95% B220+) by removing T cells with anti-Thy1.2 Ab-coated magnetic beads (Dynal, Lake Success, NY), macrophage/monocytes, dendritic cells, and NK cells with rat anti-CD43 mAb and sheep anti-rat IgG-coated magnetic beads (Dynal). Purified splenic B cells were cultured in 0.6 ml of culture medium in 48-well flat-bottom plates and stimulated with 10 or 100 μg/ml of LPS (0111:B4; Sigma-Aldrich). In other experiments, B cells were cultured with 200 or 500 μg/ml of HA (MP Biomedicals, Solon, OH), 1000 μg/ml of fibrinogen (Sigma-Aldrich), 100 μg/ml of HS (Sigma-Aldrich), 200 μg/ml of dermatan sulfate (Sigma-Aldrich), or 200 μg/ml of chondroitin sulfate (Sigma-Aldrich) for 12 hours. Anti-mouse TLR4 mAb (MTS510; BioLegend, San Diego, CA) or control rat IgG2a (R&D systems) was added 60 minutes before HA stimulation at concentrations of 100 μg/ml. In the experiments reported here, culture medium contained 95%). Purified macrophages were cultured in 0.6 ml of serum-free RPMI medium in 48-well flat-bottom plates at 37°C for 8 hours with IL-6 (Acris antibodies, Hiddenhausen, Germany), IL-10 (Acris antibodies), or TGF-β1 (R&D systems) at the indicated concentrations. Expression of bFGF was analyzed using a real-time PCR quantification method. Culture supernatants from unstimulated or stimulated macrophages were also analyzed for the production of bFGF by specific ELISA kit (R&D systems). Primary murine keratinocytes and fibroblasts were isolated from wild-type, CD19−/−, and CD19Tg mice as previously described.21Marcelo CL Kim YG Kaine JL Voorhees JJ Stratification, specialization, and proliferation of primary keratinocyte cultures. Evidence of a functioning in vitro epidermal cell system.J Cell Biol. 1978; 79: 356-370Crossref PubMed Scopus (154) Google Scholar Cells (4 × 103 cells) were cultured in 0.2 ml of Dulbecco's modified eagle medium in 96-well culture plates with or without 1 ng/ml of bFGF (Kaken Pharmaceutical, Tokyo, Japan) for 72 hours. Cellular proliferation was quantified by the addition of 10 μmol/L 5-bromo-2-deoxyuridine (BrdU; Roche Diagnostics, Mannheim, Germany) during the last 24 hours of a 72-hour culture, and BrdU incorporation was assayed by ELISA (Roche Diagnostics), according to the manufacturer's instructions. Purified splenic B cells (1 × 107 cells) from wild-type, CD19−/−, and CD19Tg were stimulated with 10 μg/ml of LPS (Sigma-Aldrich), and subsequently lysed for 30 minutes on ice in buffer containing 100 mmol/L Tris (pH7.4), 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L NaF, 20 mmol/L Na4P2O7, 2 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mmol/L PMSF, and protease inhibitors. Protein concentrations were determined by light absorbance at 280 nm. The phosphorylation level of p38 was analyzed using PhosphoDetect p38 mitogen-activated protein kinase (MAPK) (pThr180/pTyr182) ELISA kit (Calbiochem). Growth factors (bFGF and PDGF) or HA were applied to each wound in 20-μl aqueous buffer immediately and 12 hours after wounding, and wounds were covered with an occlusive dressing (Tegaderm, 3M Canada). The amounts of growth factors or HA used in this study were as follows: bFGF (Kaken Pharmaceutical), 1000 ng/20 μl; PDGF B-B isoform (AUSTRAL Biologicals, San Ramon, CA), 800 ng/20 μl; and HA (MP Biomedicals), 20 μg/20 μl. Optimal amounts of growth factors were determined elsewhere.22Mori T Kawara S Shinozaki M Hayashi N Kakinuma T Igarashi A Takigawa M Nakanishi T Takehara K Role and interaction of connective tissue growth factor with transforming growth factor-beta in persistent fibrosis: a mouse fibrosis model.J Cell Physiol. 1999; 181: 153-159Crossref PubMed Scopus (427) Google Scholar Macroscopic area of the open wound was measured at 3 and 7 days after wounding. For the analysis, 15 mice (50 wounds) in wild-type group, 14 mice (42 wounds) in CD19−/−, and CD19Tg group, and 10 mice (38 wounds) in groups of CD19−/− treated with HA or growth factors were used. The Mann-Whitney U-test was used for determining the level of significance of differences between samples, and Bonferroni's test was used for multiple comparisons. A P value <0.05 was considered statistically significant. The areas of open wounds were measured at 3 and 7 days after wounding to assess macroscopic healing defects (Figure 1, A and B). At both 3 and 7 days after injury, open wound area was larger in CD19−/− mice than that in wild-type and CD19Tg mice. However, macroscopic wound healing in CD19Tg mice was similar to that in wild-type mice. Re-epithelialization was assessed by microscopically measuring the epithelial gap that is the distance between the migrating edges of keratinocytes (Figure 1, A and C). The epithelial gap was longer in CD19−/− mice relative to wild-type mice, at both 3 and 7 days after wounding. Inversely, it was shorter in CD19Tg mice compared with wild-type mice. The area of granulation tissue was also measured microscopically (Figure 1, A and D). Granulation tissue formation was inhibited in CD19−/− mice relative to wild-type and CD19Tg mice at both 3 and 7 days after wounding. Inversely, CD19 overexpression promoted granulation tissue formation relative to wild-type mice. Thus, CD19 loss inhibited cutaneous wound healing, whereas CD19 overexpression promoted it. The number of neutrophils that migrated outside the blood vessels was reduced in CD19−/− mice relative to wild-type mice at 3 days but not 7 days after wounding (Figure 2, A and D). Neutrophil numbers in CD19Tg mice tended to be higher than those found in wild-type mice after 3 days; however, the difference did not reach statistical significance (P = 0.09). Macrophage numbers decreased in CD19−/− mice relative to wild-type mice as well as CD19Tg mice at both 3 and 7 days after injury (Figure 2, B and D). However, CD19 overexpression did not affect macrophage numbers. By contrast, there were no significant differences in numbers of mast cells, CD3+ T cells, and B220+ B cells among the different genotypes of mice at both 3 and 7 days after injury (Figure 2C and data not shown). However, infiltration of B cells in the wound tissue was confirmed (Figure 2E). There was no significant difference in B cell numbers among different wound sites (epidermal wound margin, edges of panniculus carnosus, and wound basis; Figure 2E and data not shown). The number of resident cutaneous inflammatory cells at the time of pre-wounding (day 0) did not differ significantly among the different genotypes (data not shown). Thus, CD19 deficiency reduced infiltration of neutrophils and macrophages, whereas altered CD19 expression did not affect the infiltration of mast cells, CD3+ T cells, or B220+ B cells. Expression of bFGF, aFGF, IL-6, IL-10, TGF-β, PDGF, TNF-α, and IFN-γ mRNA in wounded skin tissue was examined by real-time PCR (Figure 3). At 3 days after wounding, CD19−/− mice had decreased mRNA expression levels of bFGF, aFGF, IL-6, IL-10, PDGF, and TGF-β relative to wild-type mice and CD19Tg mice, whereas at 7 days after wounding, the significant difference between CD19−/− and wild-type mice was observed only in bFGF and aFGF mRNA levels. By contrast, CD19Tg mice had elevated levels of aFGF, PDGF, and TGF-β mRNA relative to wild-type mice and CD19−/− mice at 3 days after wounding. Although levels of bFGF, IL-6, IL-10, and TNF-α in CD19Tg mice were higher than those in CD19−/− and were also tend to be higher compared with wild-type mice, these differences did not reach statistical significance. At 7 days after wounding, bFGF, aFGF, TGF-β, and TNF-α mRNA levels were elevated in CD19Tg mice relative to CD19−/− mice, while they were similar between CD19Tg and wild-type mice. Finally, the loss or overexpression of CD19 did not affect IFN-γ mRNA expression. Thus, CD19 deficiency reduced expression of bFGF, aFGF, IL-6, IL-10, PDGF, and TGF-β, while CD19 overexpression increased expression of aFGF, PDGF, and TGF-β. Since CD19 regulates B cell response by LPS stimulation,14Engel P Zhou LJ Ord DC Sato S Koller B Tedder TF Abnormal B lymphocyte development, activation, and differentiation in mice that lack or overexpress the CD19 signal transduction molecule.Immunity. 1995; 3: 39-50Abstract Full Text PDF PubMed Scopus (492) Google Scholar, 15Sato S Steeber DA Jansen PJ Tedder TF CD19 expression levels regulate B lymphocyte development: human CD19 restores normal function in mice lacking endogenous CD19.J Immunol. 1997; 158: 4662-4669Crossref PubMed Google Scholar it is possible that CD19 affects TLR4-mediated cytokine production by B cells, resulting in the different cytokine levels within the wound tissue among the different genotypes. Therefore, we stimulated B cells with LPS and examined their cytokine mRNA expression by real-time PCR analysis (Figure 4A). Unstimulated B cells from CD19Tg mice spontaneously produced a higher mRNA amount of IL-6, IL-10, and TNF-α relative to wild-type B cells. LPS stimulation increased IL-6 mRNA expression by B cells from wild-type and mutant mice in a dose-dependent manner. Similarly, although not in a dose-dependent manner, IL-10, TGF-β, and TNF-α mRNA levels also increased by LPS stimulation. Furthermore, levels of IL-6, IL-10, and TGF-β mRNA correlated with CD19 expression levels. Consistently, double staining of B cells in the skin tissues using anti-B220 (stained brown) and IL-10 (stained green) Abs revealed that IL-10 expression was decreased in CD19−/− mice and increased in CD19Tg mice relative to wild-type mice (Figure 4B). TNF-α mRNA levels were increased by CD19 overexpression but not significantly reduced by CD19 loss, regardless of LPS stimulation (Figure 4A). None of bFGF, aFGF, and PDGF was detected in B cells even after stimulation with LPS (data not shown). Thus, LPS-induced IL-6, IL-10, and TGF-β production by B cells generally correlated with CD19 expression levels. The recent identification of endogenous ligands of TLRs, including HA, heparan sulfate (HS), and fibrinogen, indicates that they function not only to induce defensive antimicrobial immune responses, but also as a sensitive detection system to initiate tissue regeneration after injury.23Zhang Z Schluesener HJ Mammalian toll-like receptors: from endogenous ligands to tissue regeneration.Cell Mol Life Sci. 2006; 63: 2901-2907Crossref PubMed Scopus (116) Google Scholar Bot
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