Receptor Signaling Fibroblast Proliferation Involves IL-4 Synergy

Synergy Between CD40 Ligation and IL-4 on Fibroblast Proliferation Involves IL-4 Receptor Signaling

This information is current as Sergei P. Atamas, Irina G. Luzina, Heqiao Dai, Susan G. of October 1, 2021. Wilt and Barbara White J Immunol 2002; 168:1139-1145; ; doi: 10.4049/jimmunol.168.3.1139 http://www.jimmunol.org/content/168/3/1139 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Synergy Between CD40 Ligation and IL-4 on Fibroblast Proliferation Involves IL-4 Receptor Signaling1

Sergei P. Atamas,2*† Irina G. Luzina,*† Heqiao Dai,* Susan G. Wilt,† and Barbara White*†

Fibrosis can be an undesired consequence of activated cellular immune responses. The purpose of this work was to determine whether CD40 ligation and the pro-fibrotic cytokine IL-4 interact in regulating fibroblast proliferation and collagen production, and, if so, the mechanisms used. This study found that the combination of IL-4 and ligation of CD40 on the fibroblast cell surface had synergistic effects in stimulating fibroblast proliferation. In contrast, CD40 ligation negated the inhibitory effects of IFN-␥ on fibroblast proliferation. Western blotting analyses of fibroblast crude lysates revealed that a potential mechanism of the synergy between CD40 ligation and IL-4 was the phosphorylation of proteins at 130 kDa and, to a lesser degree, at 95, 85, and 75 kDa. Immunoprecipitation-Western blotting experiments showed that phosphorylation levels of IL-4R␣, Janus kinase 1, insulin receptor substrate 1, and insulin receptor substrate 2, factors with molecular mass close to the observed 130 kDa major phosphorylation band, increased in response to the combined CD40 ligation and IL-4 action. In contrast, there was no evidence that synergy Downloaded from was mediated by an increased expression of IL-4R␣ chain, CD40, or the autocrine profibrotic cytokines IL-6 and TGF-␤. These findings suggest that CD40-CD40 ligand contacts between fibroblasts and cells secreting IL-4 may promote the profibrotic effects of IL-4 by affecting signal transduction and reducing the anti-fibrotic effects of IFN-␥. The Journal of Immunology, 2002, 168: 1139–1145.

nwanted tissue damage can occur when fibrosis follows toward IL-4 in the lungs of scleroderma patients is associated with http://www.jimmunol.org/ an activated cellular immune response in that tissue. Ac- progressive interstitial lung disease (25). U tivated T cells of both Th1 and Th2 types can interact Activated T cells express CD40 ligand (CD40L)3 (26), as do with fibroblasts and regulate their activities by secreting IFN-␥ and mast cells (27), basophils (28), and eosinophils (29). CD40L binds IL-4, respectively. Mast cells also regulate activities of fibroblasts CD40, a member of the TNFR family that is expressed and func- through IL-4 secretion (1, 2), as do basophils (3) and eosinophils tionally active in a variety of cell types (30, 31). Fibroblasts from (4). IL-4 is a profibrotic cytokine. It stimulates fibroblast chemo- the skin, lung, gingiva, synovium, and spleen all express CD40 taxis, proliferation, expression of adhesion molecules ICAM-1 and (31), which implies potential interactions with T cells through CD40L-CD40 contacts. In fibroblasts, CD40 ligation stimulates VCAM-1, and production of IL-6 and extracellular matrix proteins by guest on October 1, 2021 in vitro (1, 5Ð12). Exposure to IL-4 leads to an increase in collagen proliferation (32) and has other activating effects, including mo- production in the lungs (4, 13), and correlates with the amount of bilization of NF-␬B, production of IL-6 and IL-8 (30, 32, 33), and hepatic fibrosis (14). Normal wound healing is accelerated by IL-4 stimulation of ICAM-1 and VCAM-1 expression (32). CD40 liga- (2, 15). Treatment with anti-IL-4 and the targeted mutation of IL- tion is important in the development of pulmonary fibrosis in an- 4R␣ prevent dermal fibrosis in the tight-skin mouse model of imals. Blockage of CD40L-CD40 interactions protects against ra- scleroderma (16, 17). The profibrotic effects of IL-4 on fibroblasts diation-induced pulmonary fibrosis (34) and hapten immune can be counterbalanced by IFN-␥, which inhibits fibroblast prolif- pulmonary interstitial fibrosis (35). eration and production of collagen in vitro (18Ð21), inhibits ex- The two factors, CD40 ligation and IL-4, interact synergistically perimental renal fibrosis (22), and reduces size of keloids (23). In in stimulating proliferation (36) and isotype switching, particularly a bleomycin model of lung fibrosis, inhibition of IL-4 production IgE production (37, 38), in B cells. Recently, synergy of the two by lung T cells in the setting of unchanged IFN-␥ production leads factors has been described in up-regulation of adhesion molecules to reduced lung fibrosis and prolonged survival (24). Conversely, expressed on endothelial cells (39). The goal of this work was to a shift in the balance between IL-4 and IFN-␥ mRNA production investigate whether these two profibrotic factors have synergistic effect on fibroblasts.

Materials and Methods *Department of Medicine, University of Maryland School of Medicine, and †Re- search Service, Veterans Affairs Medical Center, Baltimore, MD, 21201 Fibroblast cell lines Received for publication September 26, 2001. Accepted for publication November 21, Already existing normal human skin fibroblast lines were used. These lines 2001. were established from primary dermal explants as described previously The costs of publication of this article were defrayed in part by the payment of page (40). Fibroblast lines were maintained in T75 culture flasks in humidified charges. This article must therefore be hereby marked advertisement in accordance atmosphere of 5% CO2 at 37¡C in high serum tissue culture medium, which with 18 U.S.C. Section 1734 solely to indicate this fact. was DMEM supplemented with 2 mM glutamine, 2 mM sodium pyruvate, 1 This work was supported in part by a Research Grant from the Scleroderma Foun- 50 mg/L gentamicin, and 10% bovine calf serum (all from Life Technol- dation, National Institute of Health Grant 1R03AR47110 (to S.P.A.) and by the Na- ogies, Grand Island, NY). tional Institute of Health Grant 1R01HL54163 (to B.W.). 2 Address correspondence and reprint requests to Dr. Sergei Atamas, Division of Rheumatology and Clinical Immunology, University of Maryland School of Medi- cine, MSTF 8-34, 10 South Pine Street, Baltimore, MD 21201. E-mail address: 3 Abbreviations used in this paper: CD40L, CD40 ligand; TRAF, TNFR-associated [email protected] factor; JAK, Janus kinase; IRS, insulin receptor substrate.

Cell proliferation and collagen production assays were collected using a Storm densitometer and band densities analyzed with ImageQuant software (Molecular Dynamics). Two inhibitors of in- Fibroblast cell lines were tested in passages three to seven. They were tracellular signal transduction, genistein and PD98059, were purchased grown to confluency, detached by trypsinization (Life Technologies), from Sigma Aldrich. washed, and replated in high serum tissue culture medium in the desired test wells overnight. Two different proliferation assays were used. For use IL-6 and TGF-␤1 assays in the CellTiter AQ 96 Non-Radioactive Cell Proliferation Assay (Pro- ueous ␤ mega, Madison, WI), fibroblasts were plated at 2 ϫ 103 cells per well in ELISPOT analyses for IL-6 and TGF- 1 production by fibroblasts were 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA) in performed using Unifilter Polyfiltronics ELISPOT plates (Whatman, 0.2-ml cultures. For testing in a [3H]thymidine (Amersham Pharmacia Bio- Clifton, NJ), according to the manufacturer’s instructions. Briefly, the tech, Piscataway, NJ) incorporation assay, fibroblasts were plated at 3 ϫ wells on the ELISPOT plates were coated with capture Abs diluted in PBS ϫ 104 per well in 12-well plates (Costar) in 2-ml cultures. After overnight and then blocked with 1% BSA in PBS. Fibroblasts were cultured at 2 4 ␮ incubation in high serum tissue culture medium, the medium in each well 10 cells/ml in 100 l cultures, alone or with the desired test substances, was replaced with DMEM containing the same supplements listed above, and incubated for 2Ð5 days at 37¡C in humidified 5% CO2. Anti-IL-6 and ␤ except the serum concentration was decreased to 0.5% (low serum tissue anti-TGF- 1 capture and developing Abs were purchased from R&D Sys- culture medium). The fibroblasts were incubated for another 24 h before tems (Minneapolis, MN). Strepavidin-alkaline phosphatase conjugate (Up- adding the desired test substances. Recombinant human IL-4, IFN-␥ (both state Biotechnology) and nitro blue tetrazolium substrate (Sigma Aldrich) from Life Technologies), soluble CD40L (ALEXIS Biochemicals, San Di- were used for color development. The resulting spots were counted on a ego, CA), and mouse monoclonal anti-human CD40 (BD PharMingen, San computer-assisted image analyzer (Autoimmun Diagnostika, Beltsville, Ϯ Diego, CA) were used, alone or in combination. Low serum tissue culture MD). Data were expressed as spot counts SD per culture of quadriplicate medium alone was the negative control. The CellTiter proliferation assay cultures. was done according to the manufacturer’s instructions, after the fibroblasts Expression of cell surface molecules were incubated with test substances for 3Ð7 days. Data were expressed as

Ϯ 3 ␣ ␥ Downloaded from mean OD490 SD of quadriplicate cultures. In the [ H]thymidine incor- Expression of IL-4R , CD40, and common -chain on fibroblasts was poration assay, fibroblasts were incubated with or without test substances studied by immunofluorescence using FITC-labeled specific Abs (BD for 2Ð5 days, then [3H]thymidine was added to each culture at a final PharMingen). Fibroblasts were allowed to adhere in chamber slides (Lab- concentration of 1 mCi/ml for8hofincubation. Then, the cultures were Tek; Nalge Nunc, Naperville, IL) at 2 ϫ 104 cells/ml, preincubated over- washed twice with ice-cold PBS, five times with 5% TCA, and lysed in 0.1 night in low serum tissue culture medium, and then activated for 3 days N NaOH/0.1% SDS. A 100-␮l aliquot of each lysate was measured in a with IL-4 and soluble CD40L, or mouse monoclonal anti-CD40, alone and Beckman beta scintillation counter (Beckman Coulter, Fullerton, CA). in combinations. Cells were fixed with 2% paraformaldehyde in PBS, pH Data were expressed as cpm Ϯ SD of quadriplicate cultures. 7.4. Slides were then blocked with 5% goat serum and stained with specific http://www.jimmunol.org/ For the collagen production assay, fibroblasts were plated at 2 ϫ 105 Abs. Nuclei were stained with propidium iodide. Digital images were ac- cells/well in 6-well plates (Costar), incubated overnight in 3 ml/well of quired using a Nikon Microfot FX fluorescent microscope equipped with high serum medium, and then for 24 h in low serum medium. After that, Sony DKC CCD camera, magnification ϫ200, using uniform setting for all the culture medium was replaced with 1 ml/well of fresh low serum me- slides. Images were exported into a personal computer, and the intensity of dium with or without added test substances and containing 14C-proline at surface staining was analyzed using IPLab imaging software (Scanalytics, 1 ␮Ci/ml (Amersham Pharmacia Biotech), 280 ␮M sodium ascorbate, and Fairfax, VA). Mean density of staining was calculated for 50 cells on each 200 ␮M ␤-aminopropionitrile (both from Sigma Aldrich, St. Louis, MO). slide and data were compared using a t test. After 24 h, cell culture supernatants were collected, rapidly frozen in liquid nitrogen and freeze-dried at Ϫ70¡C. The pellets were dissolved in 100 ␮l Results Laemmli buffer per 1 ml of the cell culture supernatant, and the samples Synergistic stimulation of fibroblast proliferation by CD40 were electrophoretically separated in 7.5% acrylamide gels. Fluorographic ligation and IL-4 by guest on October 1, 2021 images were developed using EN[3H]ANCE autoragiography enhancer (NEN, Boston, MA). Gel images were collected using Storm densitometer Fibroblast proliferation was up-regulated by IL-4 in a dose-depen- (Molecular Dynamics, Sunnyvale, CA), and band densities were analyzed dent fashion from 1 to 100 U/ml, with maximum increase in pro- with ImageQuant software (Molecular Dynamics). The identity of two major collagen bands was confirmed by sensitivity to collagenase (Sigma liferation of 2.5-fold. Recombinant human soluble CD40L and anti- Aldrich) digestion. CD40 mAb used from 0.01 to 1 ␮g/ml caused a dose-dependent Differences in fibroblast response to stimulation were evaluated using increase in proliferation, with maximum increase of 2-fold. rIFN-␥ two-tailed t test. To confirm synergy, the observed response to the com- inhibited proliferation 1.3-fold at 500 U/ml and 1.7-fold at 1000 bined factors was compared with the response to be expected based on the U/ml. These fold-changes in proliferation are consistent with re- additive action of the factors. ports previously published by others (7, 19, 32). Based on the dose-dependence data for the factors alone, suboptimal concentra- Immunoblotting for tyrosine phosphorylation of signal tions of the factors were chosen for the experiments with the fac- transduction factors tors combined to avoid saturation of the proliferative response by Fibroblasts were plated in 6-well plates (Costar) at 2 ϫ 105 cells per well each factor alone. In combinations, IL-4 was used at 10 U/ml, in 3-ml culture in high serum tissue culture medium overnight. Then, the anti-CD40 mAb and CD40L were used at 0.1 ␮g/ml, and IFN-␥ medium was replaced with low serum medium and fibroblasts incubated for another 24 h. After incubation with IL-4 and CD40L for 1Ð30 min, was used at 500 U/ml. Incubation of fibroblasts with IL-4 com- fibroblast cultures were washed with ice-cold PBS containing 100 ␮M bined with either anti-CD40 mAb or CD40L caused an increase in Na3VO4. Fibroblasts were lysed with 1 ml ice-cold lysis buffer containing proliferation with the amplitude that was higher than a combina- 0.5% Nonidet P-40, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, and 10% tion of independent effects of IL-4 alone and anti-CD40 mAb or protease inhibitor mixture (Sigma Aldrich) in PBS for immunoprecipitation, or were lysed in 250 ␮l of Laemmli sample buffer for analysis of CD40L alone (Fig. 1, A and B provide two examples). Five fibro- overall pattern of tyrosine phosphorylation. For immunoprecipitation, the blast cell lines derived from the skin of different healthy individ- lysates were incubated with 1 ␮g the desired Ab for1honice. Immune uals were studied in five or more independent experiments with complexes were precipitated with protein A-Sepharose beads (Amersham each cell line. Two lines (NDF1 and NDF2) consistently demon- Pharmacia Biotech), and eluted by 5 min boiling in Laemmli sample buffer. strated synergistic up-regulation of proliferation in response to Electrophoretic separation of immunoprecipitated proteins or cell lysates was done in 7.5% acrylamide gels, and bands were transferred onto Im- combined CD40 ligation and IL-4, with the effect of the combi- mobilon NC membranes (Millipore, Bedford, MA). Membranes were nation greater than the sum of the independent effects ( p Ͻ 0.01 in probed with specific Abs at 1/200 dilution and visualized with an ECL all cases). These two lines were used in the subsequent experi- detection system (Pierce, Rockford, IL) that was used according to the ments addressing the possible mechanism of synergy. In two more manufacturer’s directions. Anti-phosphotyrosine mAb (4G10) and Abs against IRS-1 and IRS-2 were purchased from Upstate Biotechnology lines, a tendency toward synergistic response was seen in all ex- (Lake Placid, NY). Abs against IL-4R␣, JAK1, JAK3, and gp130 were periments, although the difference between the calculated additive purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Gel images response and observed synergistic response only reached statistical The Journal of Immunology 1141

IL-4, with a maximum 3-fold excess in secreted collagen. In contrast, ligation of CD40 caused little increase in collagen production (0.9- to 1.2-fold, p Ͼ 0.05). No synergy between the two factors on collagen production was observed in any of the ﬁve lines studied; the effect of the factors combined did not exceed the effect of IL-4 alone in more than one experiment for any line (data not shown).

Changes in receptor expression and autocrine cytokine production are unlikely mechanisms of the synergy The interactions between CD40 ligation and IL-4 could be mediated through up-regulation in IL-4R and/or CD40 expression. In two experiments, each with two cell lines, a 2- to 3-fold up-regulation vs control ( p Ͻ 0.05) of IL-4R␣ was detected by immu- noﬂuorescent staining after stimulation with IL-4, whereas CD40 ligation had no effect on IL-4R␣ expression (1.0- to 1.3-fold stimulation, p Ͼ 0.05), and the combined action of the two factors on IL-4R␣ expression was not different from the effect of IL-4 alone (0.8- to 1.2-fold difference, p Ͼ 0.05; example shown in Fig. 2).

Similarly, CD40 expression was not increased by IL-4 or CD40 Downloaded from ligation, alone or in combinations ( p Ͼ 0.05 for all comparisons; data not shown), confirming earlier reports (31). This lack of synergistic increase in IL-4R␣ and CD40 expression was confirmed by Western blotting of fibroblast lysates for these proteins (data ␣ FIGURE 1. Fibroblast proliferation in response to IL-4, IFN-␥, CD40 not shown). Thus, up-regulation in IL-4R or CD40 expression ligation, and their combinations. Proliferation assays were performed after http://www.jimmunol.org/ 7 days of stimulation with the cytokines and anti-CD40 mAb, using the Ϯ colorimetric CellTiter AQueous assay. Data are given as the mean OD490 SD of quadruplicate cell cultures. A and B, Two different cell lines, NDF1 and NDF2, respectively, stimulated with IL-4 or anti-CD40 mAb alone or in combination. The observed increase in proliferation caused by the combination was higher than the calculated additive effects of CD40 ligation and IL-4 alone (p Ͻ 0.01 for both cell lines). C, Fibroblasts were cultured in the medium containing IFN-␥ or anti-CD40 mAb alone or combined. CD40 ligation neutralized the inhibitory effect of IFN-␥. D, Fibroblasts were cultured with IFN-␥ alone or in combinations with either anti-CD40 by guest on October 1, 2021 mAb or IL-4, or both factors combined. CD40 ligation and IL-4 alone only partially neutralized the inhibitory effect of IFN-␥, whereas the combination of these two factors taken at the same concentrations changed inhibition to stimulation. Cells incubated in cell culture medium with no addi- tives were used as controls. significance ( p Ͻ 0.05) in some experiments. In one line, the proliferative response to the combined CD40 ligation and IL-4 was not synergistic. Although the fibroblast lines demonstrated variability in levels of CD40 and IL-4R␣ cell surface expression, as revealed by im- munohistochemical staining, the differences among the lines in response to the combined CD40 ligation and IL-4 did not correlate with the levels of expression of these receptors. All of the studied lines responded to CD40 ligation and IL-4 alone; although the degree of response varied among the lines, the heterogeneity in responsiveness did not correlate with level of the surface receptors (data not shown). Incubation of fibroblasts with IFN-␥ caused inhibition of proliferation, which could be neutralized by CD40 ligation (Fig. 1C). Anti-CD40 Ab and IL-4 alone at chosen concentrations partially neutralized the inhibitive action of IFN-␥, whereas a combination of these two factors taken at the same concentrations reversed the FIGURE 2. Immunofluorescent staining with FITC-labeled Ab for IL- 4R␣ on human dermal fibroblasts (NDF1). Cells were incubated for 3 days overall effect from inhibition to stimulation (Fig. 1D). Thus, CD40 ␥ in cell culture medium (A), IL-4 (B), anti-CD40 mAb (C), and their com- ligation can shift the effects of IL-4 and IFN- toward the prolif- bination (D). Green fluorescence channel data were electronically con- erative action of IL-4 and against the anti-proliferative action of verted to a gray scale and inverted. The mean Ϯ SD intensities of cell IFN-␥. surface staining (arbitrary units quantified with IPLab imaging software) Stimulation of the fibroblast lines in a similar fashion led to a for AÐD were: 27.5 Ϯ 15.2, 78.3 Ϯ 27.1, 32.4 Ϯ 18.7, and 72.1 Ϯ 17.7, dose-dependent increase in collagen production in response to respectively. 1142 CD40 LIGATION AND IL-4 SYNERGIZE TO STIMULATE FIBROBLASTS was unlikely to be a mechanism of the synergistic CD40 ligation- IL-4 action on fibroblasts. Another potential mechanism of CD40 ligation-IL-4 synergy could involve an increased production of autocrine factors that stimulate fibroblast proliferation. Two cytokines have been impli- cated as autocrine profibrotic factors, TGF-␤ (e.g., Ref. 41) and IL-6 (7, 42, 43). Despite antagonistic action of IL-4 and IFN-␥ on fibroblasts, ELISPOT experiments in both studied cell lines showed that both cytokines in tested concentrations up-regulated production of IL-6 by fibroblasts ϳ1.5- to 2-fold ( p Ͻ 0.05), as did FIGURE 4. Western blotting analysis of phosphorylation patterns in anti-CD40 mAb (Fig. 3), which is consistent with published data NDF1 fibroblasts. Electrophoretically separated fibroblast crude lysates for all three factors (7, 32, 44). There was no synergy of anti-CD40 were stained using anti-phosphotyrosine Abs (A and B) and anti-gp130 Ab mAb or CD40L with either cytokine on stimulated IL-6 production (C). A, From left to right, fibroblasts were activated for 12 min with culture (the range of the effect was a 0.8- to 1.3-fold stimulation in com- medium, IL-4, CD40L, and IL-4 and CD40L combined. B, Fibroblasts parison with either IL-4 or IFN-␥ alone, p Ͼ 0.05, data not shown). were pre-stimulated for 72 h with CD40L, 0.1 ␮g/ml, and then activated as Production of another autocrine cytokine, TGF-␤1, was at the low in A. C, Equal sample loading for A was confirmed by staining with anti- threshold of detection in control cultures, with the positive control gp130 Ab. Equal loading in B was confirmed in the same manner (data not of PBMC showing high levels of TGF-␤1 producing cells. Pro- shown). Neither IL-4 nor CD40 ligation have been reported to affect the duction of TGF-␤1 was not affected by either CD40 ligation or expression of gp130 protein in fibroblasts. Downloaded from IL-4, alone or in combinations (data not shown). Thus, the observed combined effects were unlikely to be explained by an autocrine cytokine mechanism involving IL-6 or TGF-␤1. to the combined CD40L and IL-4, immunoprecipitation-Western blotting experiments were performed. TNFR-associated factor Protein tyrosine kinase signaling in fibroblasts is synergistically (TRAF)-2, TRAF-3, TRAF-5, and TRAF-6, which are main sig- activated by CD40 ligation and IL-4 naling molecules from CD40, were not considered as potential http://www.jimmunol.org/ The CD40 ligation and IL-4 synergy could be mediated by intra- contributors to the 130 kDa phosphorylation band because of their cellular signaling. Western blotting of electrophoretically sepa- lower molecular mass of ϳ55Ð65 kDa (reviewed in Ref. 45). In rated crude lysate of nonactivated fibroblasts revealed a scarce contrast, molecules participating in intracellular signaling from pattern of constitutive protein tyrosine phosphorylation, with one IL-4, such as IL-4R, Janus kinase (JAK1), insulin receptor sub- major band at ϳ130 kDa. The level of protein tyrosine phosphor- strate (IRS1), and IRS2, have molecular mass of ϳ130 kDa (46). ylation in this band was synergistically up-regulated by fibroblast Phosphorylation of IL-4R␣, JAK1, IRS1, and IRS2 in fibroblasts exposure to CD40L and IL-4 (Fig. 4A). Preincubation with CD40L was synergistically up-regulated in response to the combined for 3 days did not change the background pattern of protein ty- CD40L and IL-4, whereas phosphorylation of an irrelevant cyto-

rosine phosphorylation, but affected the pattern of phosphorylation kine receptor chain, gp130, did not change (Fig. 5). JAK3 was not by guest on October 1, 2021 after fibroblast exposure to combined CD40L and IL-4, with pre- detected in the studied fibroblast lines (data not shown). These served synergy on the level of phosphorylation in the 130 kDa findings suggest that CD40 ligation and IL-4 synergy on fibroblast band, and additional bands at ϳ95, 85, and 75 kDa (Fig. 4B). proliferation may be mediated through changes in IL-4 protein Preincubation with IL-4 did not cause such a change in tyrosine tyrosine kinase signaling. phosphorylation. Although preincubation with CD40L increased To confirm that tyrosine kinase signaling was involved in this the amplitude of phosphorylation in response to the combination of synergy, fibroblasts were stimulated with anti-CD40 mAb, IL-4, or CD40 ligation and IL-4, it had little, if any, effect on the amplitude the combination, with or without two pharmacologic inhibitors of of the proliferative response of fibroblasts to IL-4 or CD40L, either intracellular signaling. Genistein, an inhibitor of protein tyrosine alone or combined. kinases, and PD98059, an inhibitor of mitogen-activated protein To identify specific molecules contributing to increased phos- (MAP) kinase kinase, were both used at 30 ␮M concentration. phorylation in the major 130 kDa elctrophoretic band in response Genistein caused 65Ð70% inhibition of fibroblast proliferation, whereas PD98059 was less effective, causing only 25Ð30% inhibition (data not shown). Thus, it appears that tyrosine phosphorylation was involved in signaling from CD40 and IL-4.

Discussion Synergy of CD40 ligation and IL-4 in the regulation of B cell proliferation and isotype switching is well established (36Ð38). Similarly, these two factors synergize in up-regulation of adhesion molecule expression on endothelial cells (39). This work demon- strates that such synergy extends to fibroblasts (Fig. 1). Synergistic up-regulation of fibroblast proliferation by CD40 ligation and IL-4 could be mediated by T cells, mast cells, eosinophils, and basophils, all of which can express CD40L and secrete IL-4. Such synergy can potentially contribute to regulation of fibroblast activities in health and pathology, such as normal wound healing, the FIGURE 3. IL-6 production by NDF1 fibroblast cultures. Cells were formation of hypertrophic scars (2), and the development of tissue stimulated with IL-4, IFN-␥, anti-CD40 mAb, and IL-4 plus anti-CD40 fibrosis following inflammation (25). mAb combination for 2 days. Data are shown as quadruplicate mean counts The stimulating effect of IL-4 on fibroblast proliferation can be of IL-6 producing cells Ϯ SD, as detected by ELISPOT assay. opposed by an inhibitory action of IFN-␥ (Fig. 1). In vivo, both The Journal of Immunology 1143

ergistically increase phosphorylation of several proteins, with a particularly strong band at 130 kDa. To identify proteins that might contribute to that band, molecules known to be involved in IL-4R or CD40 signaling were considered. Many of IL-4R signaling pathway molecules have a molecular mass close to 130 kDa, including JAK1, IRS1, IRS2, and IL-4R␣ itself (49Ð51). In contrast, CD40 signaling molecules, TRAFs, have lower molecular mass of 55Ð65 kDa (reviewed in Ref. 45). Of note, IL-4R signaling through IRS1 and IRS2 molecules activates proliferation (50, 51), whereas signaling through STAT6 (calculated molecular mass, 93 kDa), leads to an increase in gene translation, but not proliferation. Immunoprecipitation of fibroblast lysates with Abs against IL- 4R␣, JAK1, IRS1, and IRS2 and subsequent Western blotting for tyrosine phosphorylation demonstrated that phosphorylation of each of these factors is synergistically up-regulated by CD40 ligation and IL-4 (Fig. 5). Thus, CD40 ligation increases signaling through the IL-4R pathway in fibroblasts, leading to synergistic effect of CD40 ligation and IL-4 on fibroblast proliferation. Some aspects of IL-4R signaling in fibroblasts appear to be dif- Downloaded from ferent from that in hematopoetic cells, including B cells. Particu- larly interesting is the observation in this report of a synergistic increase in IL-4R␣ phosphorylation after stimulation of fibroblasts with combined CD40L and IL-4 (Fig. 5). In contrast to B cells (47), this effect is not caused by stimulation of IL-4R␣ expression by CD40 ligation (Fig. 2). It is possible that ligation of CD40 increases IL-4R sensitivity without increasing its level of expres- http://www.jimmunol.org/ sion, similar to the effects of CD28 ligation on Th2 cells (52). Also indicative of differences in IL-4 signaling between fibroblasts and hematopoetic cells is that JAK3 appears to be involved in CD40 ligation-IL-4 synergy in B cells, with an increase in its expression and a change in phosphorylation/dephosphorylation kinetics induced by CD40 ligation (47, 48). However, this work found that FIGURE 5. Tyrosine phosphorylation of IL-4R␣, JAK1, IRS1, IRS2, neither JAK3 nor common ␥-chain (our unpublished data), with and gp130 in fibroblasts. NDF1 cells were stimulated as described in Fig.

which JAK3 is associated in IL-4R signaling, are expressed in the by guest on October 1, 2021 4. A, Fibroblast lysates were immunoprecipitated with Abs against indi- dermal fibroblasts. Additional differences in IL-4R signaling be- cated proteins, and Western blotting developed using antiphosphotyrosine tween fibroblasts and hematopoetic cells include involvement of Ab. B, Densities of the bands from A were measured and normalized to the density of the band in the presence of combined CD40L and IL-4 set at STAT1 and STAT3 in fibroblast signaling (53), two factors con- ␥ 100%. Equal loading for each factor was confirmed by staining with Abs ventionally associated, respectively, with IFN- and IL-6/IL-10. used for immunoprecipitation (not shown). Unlike its effects in hematopoetic cells, IL-4 increases phosphorylation and activity of JNK and extracellular signal-regulated kinases in fibroblasts (54). Finally, IL-4 stimulation may be inhibited IL-4 and IFN-␥ may coexist in healthy tissues and at sites of in- by a naturally occurring splice variant, IL-4␦2 (55), in T cells (56), flammation. A shift in the ratio of IL-4 and IFN-␥ toward more B cells, and monocytes (57), whereas IL-4 and IL-4␦2 both stim- IL-4 can be associated with increased fibrosis (e.g., in Ref. 25). It ulate fibroblasts to increase collagen mRNA production (25). Fur- appears, based on the experiments reported in this study, that ther investigation is necessary to address the effects of CD40 li- CD40 ligation on fibroblasts may shift the functional balance be- gation on the consequences of fibroblast exposure to IL-4 and the tween IL-4 and IFN-␥ toward the effects of IL-4, thus promoting mechanisms by which these effects occur. fibrosis without changing the actual ratio of IL-4 to IFN-␥. The contribution of autocrine regulation of fibroblast prolifera- In B cells, synergy between CD40 ligation and IL-4 is mediated tion was tested as another possible mechanism of synergy between through up-regulation of expression of surface molecules, such as CD40 ligation and IL-4. Autocrine TGF-␤ is believed to be an IL-4R␣, and changes in signaling, particularly the expression and important regulator of fibroblast proliferation (41, 58). There was phosphorylation of JAK3 (47, 48). This report shows that changes very low production of TGF-␤1 in these fibroblast lines, and it was in expression of IL-4R␣ or CD40 do not mediate the synergy be- not stimulated by CD40 ligation, IL-4, or combinations thereof. tween CD40 ligation and IL-4 on fibroblast proliferation. Indeed, Another potential autocrine fibroblast regulator is IL-6. IL-4 stim- this work confirms the previously reported observation that expo- ulates production of IL-6 by fibroblasts (7), and IL-6 may stimulate sure to IL-4 does not increase the level of CD40 expression on fibroblast proliferation (42, 43). In B cells and endothelial cells, fibroblasts (31). In addition, this work shows that although expo- CD40 ligation and IL-4 synergistically increase production of IL-6 sure to IL-4 increases expression of IL-4R␣ on fibroblasts, CD40 (37, 39). However, autocrine IL-6 production does not appear to be ligation does not exert such an effect, nor does CD40 ligation syn- involved in this synergistic response, because both IL-4 and IFN-␥ ergize with IL-4 in up-regulating IL-4R␣ expression (Fig. 2). increase production of IL-6 by fibroblasts (Fig. 3), confirming pre- To address the possibility that changes in signaling contribute to vious reports (7, 44), although they have opposite effects on fibro- the synergy between CD40 ligation and IL-4, tyrosine phosphor- blast proliferation (Fig. 1). There was no synergy detected by ylation of signaling molecules was tested in fibroblast lysates (Fig. ELISPOT between CD40L and IL-4 in up-regulating IL-6 produc- 4). These experiments revealed that CD40 ligation and IL-4 syn- tion in NDF1 and NDF2 lines under the studied conditions. 1144 CD40 LIGATION AND IL-4 SYNERGIZE TO STIMULATE FIBROBLASTS

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