CLINICAL
IMMUNOLOI;~
.ZND
IMMUK;oPATHt~LOG\1
Vol. 63, No. 2. May. pp. 182-187. 1992
Human Recombinant Interleukin-4 Induces Proliferation and Interleukin-6 Production by Cultured Human Skin Fibroblasts CAROL A. FEGHALI,* KENNETH *Department
L. BOST,* DENNISW. BOULWARE,~ AND LAURA S.LEVY*-'
and Immunology
of Microbiology
New
and fDepartment of MedLcme, Orleans, Louisiana 70112
The effect of human recombinant interleukin-4 (hrIL-4) on normal human adult dermal fibroblasts in terms of proliferation and IL-6 production was studied. Fibroblasts were exposedto different concentrations of IL-4 for various periods of time. Proliferation was measuredusing a [3H]thymidine incorporation assay. IL-6 production was measured at the transcriptional, protein, and functional levels by Northern blot analysis, radioimmunoassay, and B9 bioassay, respectively. Our results show that hrIL-4 significantly stimulated (two- to fivefold) fibroblasts to increase the incorporation of 13H]thymidine in a dose- and time-dependent manner. However, hrIL-1, hrIL-2, hrIL-5, or hrTNFa, at the sameconcentration (100 U/ml) and for the sametime period (4 days), did not. In addition, IL-4 significantly induced (four- to eightfold) the production of immunoreactive and biologically functional IL-6 However, IL-4 was not as potent an inducer of IL-6 as IL-l. The IL-4-induced IL-6 production was dose and time dependent and was due, at least in part, to a dramatic increase in the steady-state levels of IL-6 mRNA. This is the first report describing the ability of IL-4 to activate human dermal fibroblasts in terms of proliferation and IL-6 production. E mszAcademic PRSS, IW. INTRODUCTION
Interleukin-6 (IL-61 is a cytokine that displays several biological activities. It is produced by a broad range of cells, namely B and T cells, monocytes, keratinocytes, fibroblasts, endothelial cells, and some tumor cells (1). Cell-specific expression of IL-6 results in a multitude of biological responses, including stimulation of B lymphocyte maturation into plasma cells, increase in the production of immunoglobulins, and stimulation of T cell growth and differentiation [reviewed in Refs. (241. In fact, levels of IL-6 have been reported to be elevated during infection, malignancy, autoimmunity, injury, and inflammation [reviewed in Refs.
Tulane
Unlverslty
School
of Meduxne.
fied as a B cell stimulatory factor (91, IL-4 is a lymphokine product of antigen-stimulated helper T cells and has numerous immunoregulatory effects of a stimulatory and inhibitory nature [reviewed in Refs. (3, 10, ll)]. IL-4 exerts its effects on cells that express the IL-4 receptor such as B and T lymphocytes, hematopoietic precursors, fibroblasts, epithelial cells, and endothelial cells (12). Such broad distribution of the IL-4 receptor on cells of various lineages is partly responsible for this cytokine’s diverse effects. The activities of IL-4 include the stimulation of B cell proliferation and differentiation, enhancement of IgG and IgE production, stimulation of thymocyte proliferation, stimulation of T cells (13), and stimulation of bone marrow cell growth (14). IL-4 has also been shown to affect the production of other cytokines (15-18). IL-4 is reported to downregulate IL-6 production by human peripheral blood mononuclear cells (PBMC) (191, but to upregulate IL-6 production in resting human B lymphocytes (201, in keratinocytes (21), and in endothelial cells (221. Little is known about the effect of IL-4 on human fibroblasts, although one report has shown that murine IL-4 can stimulate the proliferation of primary and immortalized murine fibroblasts (23). Another report documents the induction of foreskin fibroblast chemotaxis by human recombinant IL-4 (hrIL-41 (24). We sought to study the effects of IL-4 on human derma1 fibroblasts, particularly with respect to fibroblast proliferation and the production of fibroblast cytokines. We report that exposure to IL-4 stimulates derma1 fibroblasts to proliferate and to produce IL-6. These findings are significant because they suggest a role for IL-4 in activating dermal fibroblasts and in inducing production of IL-6 during inflammation and in certain autoimmune diseases. MATERIALS
AND METHODS
(6-811. IL-4 is also a multifunctional 1 To whom correspondence dressed.
Cells and Culture Reagents
cytokine. First identi-
and reprint
Normal adult dermal fibroblasts from a 22-year-old female were purchased from American Type Culture Collection (ATCC; No. CRL 1477) in passage 4. Cells
requests should be ad182
0090-1229/92
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
EFFECTS OF IL-4 ON HUMAN
were maintained in Dulbecco’s modified Eagle medium (D-MEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml>, streptomycin (100 pg/ml), and amphotericin B (0.25 kg/ml) at 37°C in a humidified atmosphere of 5% COs. Human recombinant IL-18, IL2, IL-4, IL-5, and tumor necrosis factor (Y (TNFa) used in this study were purchased from Amgen Biologicals, Thousand Oaks, California, and had specific activities of 5 X lo8 U/mg, 7 X 10’ U/mg, 2 X 10’ U/mg, 1 X lo6 U/mg, and 2 x lo7 U/mg, respectively. Measurement of Fibroblast Proliferation
Normal adult dermal fibroblasts growing in 25-cm2 tissue culture flasks were trypsinized and deposited in 96-well tissue culture plates at a density of 2000 cells/well. The cells were allowed to adhere for 24 hr. The medium was then replaced by D-MEM supplemented with 10% fetal bovine serum and antibiotic/antimycotic agents. IL-18, IL-2, IL-4, IL-5, or TNFa was added to the test wells, and cells were incubated for 1 to 5 days as described for each experiment. Following a 24-hr pulse with 13Hlthymidine (5 l&i/ml; 20 Ci/mmol, New England Nuclear), the cells were harvested and macromolecular DNA was collected onto Mash II glass fiber filters (Whittaker M.A. Bioproducts). The amount of radiolabel incorporated into DNA was determined by liquid scintillation counting. Measurement of IL-6 Concentration
The levels of immunoreactive Radioimmunoassay. IL-6 in culture supernatants were quantified in duplicate using a radioimmunoassay (Advanced Magnetics, Inc.) as described (25). In this assay, the lower limit of detection of IL-6 is 310 pg/ml.
branes (Nytran, Schleicher & Schuell). The membranes were then hybridized as described (28) to a 32Pradiolabeled probe prepared from the cDNA of human IL-6 (pCSF309, ATCC No. 67153). Following hybridization, membranes were washed as previously described (28) and exposed at -70°C to Kodak XRP-5 film with intensifying screens. Statistical Analysis
The data were analyzed by Student’s t test. A cutoff value of ~0.05 was used to indicate statistical significance. RESULTS
Effect of ZL-4 on Fibroblast Proliferation Effect of dose on proliferation. The proliferation of human dermal fibroblasts cultured in medium supplemented with 0, 30, 100, or 300 U/ml hrIL-4 was measured by 13Hlthymidine incorporation assay. The effect of hrIL-4 on fibroblast proliferation was measured after exposure to varying concentrations for 5 days (Fig. 1). The results show that stimulation of fibroblast proliferation by IL-4 is dose dependent, increasing with increasing IL-4 between 30 and 300 U/ml. The proliferative influence of IL-4 at 100 U/ml was compared to that of other cytokines, specifically IL-l, IL-2, IL-5, or TNFd, when added at the same concentration for the same period of time. The results show that exposure to IL-4 at 100 U/ml for 4 days results in a 2.5-fold stimulation of 13Hlthymidine incorporation, but exposure to other cytokines at the same concentration for the same time period had no stimulatory effect (Fig. 2).
B9 hybridomu proliferation assay. The levels of biologically active IL-6 in culture supernatants were quantified in a specific bioassay using the IL-6dependent murine hybridoma clone B9 (26) as described (25). To confirm the specificity of this assay as reported by others (26), we employed a monoclonal anti-IL-6 antibody (R & D Systems) and observed complete inhibition of B9 cell proliferation in its presence (data not shown). Measurement of IL-6 mRNA Levels
Confluent 25-cm2 tissue culture flasks of normal adult dermal fibroblasts in passage 12 were trypsinized and split 1:3. The medium was removed 24 hr later and replaced by fresh medium supplemented with 100 U/ml IL-l, IL-2, IL-4, or IL-5. As a control, parallel flasks were cultured without the addition of cytokine supplements. Total RNA was prepared from the flasks following the method of Chomczynski and Sacchi (27). The RNA was subjected to electrophoresis in agarose/formaldehyde gels and transferred to nylon mem-
183
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1000
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2000
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100
300
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hrlL-4 (U/ml) FIG. 1. Proliferation of fibroblasts in response to varying concentrations of hrIL4. Fibroblasts in passage 11 were seeded in 96-well tissue culture plates at a density of 2000 cells/well. Twenty-four hours later, the medium was replaced by 0.2 ml fresh medium supplemented with 0, 30, 100, or 300 U/ml hrIL-4. Following a 4-day incubation, cells were pulsed for 24 hr with [3H1thymidine (1 l&i/well) and then harvested. Macromolecular DNA was collected onto glass fiber filters, and the amount of 13H1thymidine incorporated into DNA was measured by liquid scintillation counting. Open bars represent the mean of 12 independent replicates. Shaded bars represent SEM.
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4500 4000 3500 3000 2500 2000 1500 IO00 500
E r,
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IL-1
IL-2
IL.-4
IL-5
TNFu
I
Culture
conditions
FIG. 2. Proliferation of fibroblasts in response to other cytokines. Fibroblasts in passage 11 were used as in the legend for Fig. 1, except that test media were supplemented with 100 U/ml IL-l, IL-2, IL-4, IL+, or TNFol, and cells were incubated for 3 days before a 24-hr pulse with [3H]thymidine. Shaded bars represent the mean of 12 independent replicates. Black bars represent SEM.
Kinetics
of IL4-Induced
Fibroblast
Proliferation
The kinetics of IL-4-induced proliferation was measured in fibroblasts cultured with 100 U/ml hrIL-4. As a negative control, cells were cultured in parallel in medium without the addition of cytokine. Cells were harvested at 24-hr intervals for 5 days, following a 24-hr pulse with [3Hlthymidine (1 &i/well) before each harvest. As shown in Fig. 3, the results demonstrate that fibroblasts exposed to rIL-4 (100 U/ml) over a period of 5 days respond by significantly increasing the incorporation of [3Hlthymidine relative to control cells (P s 0.05 at Days 3-5). The observed stimulation of fibroblast proliferation is time dependent, gradually increasing over a period of 5 days, whereas proliferation decreases in untreated cells as they reach confluence. Effect of IL4
on IL-6 Production
The production of IL-6 by normal human dermal fibroblasts was measured following exposure to hrIL-4
ET ;\I
or fresh medium supplemented with 100 U/ml of IL-l, IL-2, IL4, or IL-5 for a period of 4 days. Total RNA was then prepared and used in Northern blot analysis. A representative blot, shown in Fig. 6, illustrates that hrIL-4, like hrIL-1, induces significant accumulation of IL-6 mRNA in human dermal fibroblasts (three- to fourfold). IL-4 is not as potent an inducer of IL-6 mRNA as hrIL-1. No alteration in the level of IL-6 mRNA is seen following exposure to hrIL-2 or hrIL-5. These results indicate that the ability of hrIL-4 to induce IL-6 production is due, at least in part, to an accumulation of IL-6 mRNA in human dermal fibroblasts.
1.3 Kb-
FIG. 6. Effect of IL-4 on IL-6 mRNA levels. Fibroblasts were incubated in the presence of 100 U/ml IL-l, IL-2, IL-4, or IL-5 for a period of 4 days. Negative control fibroblasts were maintained in culture medium only, without the addition of any cytokine. Total RNA was prepared and used in Northern blot analysis. Equal amounts of total RNA were loaded in each lane and confirmed by ethidium bromide staining. The experiment was repeated three times.
The essential role played by interleukin-6 in the cytokine network is a subject of intense study. IL-6 serves as an important mediator of the host response to bacterial and viral infection, injury, and malignancy Ireviewed in Refs. (6-8>]. However, like other cytokines, IL-6 does not function in isolation. Rather, it is involved in a complex network of cytokine interactions within which IL-6 exerts its effect on a variety of cells and tissues. The expression of IL-6 is regulated by other cytokines such as interferons, platelet-derived growth factor, epidermal growth factor, IL-l, and IL-2 (6, 10). IL-6 gene expression can be downregulated or upregulated by various cytokines in a cell-type and tissue-specific manner. For example, IL-6 is produced at low levels by unstimulated PBMC (331, and its production is not increased by TNF (34). In contrast, TNF stimulates IL-6 gene expression in fibroblasts (31,35). Further, IL-6 production is downregulated by IL-4 in human PBMC (191, but it is upregulated by the same cytokine in B lymphocytes (201, keratinocytes (21), and endothelial cells (22). Our results show that the secretion of biologically active IL-6 is stimulated in adult human dermal fibroblasts when they are exposed to recombinant IL-4 (Figs. 4 and 5). The stimulation of IL-6 production results, at least in part, from a dramatic accumulation of IL-6 mRNA in dermal fibroblasts exposed to IL-4 (Fig. 6). We report further that IL-4 stimulates the proliferation of human dermal fibroblasts (Figs. 1, 2, and 3). Enhancement of IL-6 production and of fibroblast proliferation by IL-4 is not immediate, and more than 24 hr are necessary for the stimulatory effect to demonstrate statistical significance (P s 0.05). Such a delayed response suggests that an intermediate molecule(s) is produced to mediate the stimulatory effect of IL-4. One possibility for such an intermediate is IL-l, since IL-l is known to be produced by fibroblasts (36), and since it is a potent stimulator of IL-6 (3, 31, 35, 37-39). It is not known whether the production of IL-l by fibroblasts is influenced by IL-4. The role of IL-4 in stimulating IL-6 may be directly relevant in uivo. In scleroderma, an autoimmune connective tissue disorder of the skin and internal organs, mononuclear leukocytes typically infiltrate sites of inflammation. Cellular infiltrates in the skin consist mainly of activated T cells with a helper-inducer phenotype (40, 41). IL-4 is known to be a product of activated T helper cells; thus, IL-4 may be implicated directly in the pathogenesis of scleroderma, particularly as a stimulator of IL-6 production by fibroblasts in affected sites. In fact, we have recently shown that scleroderma fibroblasts produce as much as 30 times more IL-6 than do unaffected fibroblasts from the same patients or from a normal donor (25). Further, scleroderma patients exhibit an elevated ratio of circulating T helper to T suppressor cells (42) as well as increased
186
FEGHALI
T helper activity (43). Recently, IL-4 levels were found to be elevated in supernatants of cultured PBMC from scleroderma patients (441, and detectable IL-4 was reported more frequently in serum from scleroderma patients than from normal donors (45). IL-6 has been implicated in the pathogenesis of other autoimmune disorders as well. IL-6 levels are elevated in the sera of systemic lupus erythematosus patients (461, in the synovial fluid of patients with rheumatoid arthritis (47), in the sera of polymyalgia rheumatica and giant cell arteritis patients (481, and in psoriasis (49). Among the consequences of elevated IL-6 may be the polyclonal B cell activation and the hypergammaglobulinemia observed in inflammatory and autoimmune conditions like scleroderma and others. The data presented here represent the first report of IL-4-induced proliferation, secretion of IL-6, and increase in IL-6 mRNA by human adult dermal fibroblasts. Such stimulatory effects may indeed have a direct role in the pathogenesis of scleroderma, in which the activation of dermal fibroblasts is associated with debilitating fibrosis. IL-4 is further implicated by these data in the pathogenesis of other inflammatory and autoimmune diseases in which an elevation in IL-6 production is observed. ACKNOWLEDGMENTS The authors acknowledge Advanced Magnetics, Inc., who provided radioimmunoassay reagents for the detection of IL-6, and the American Type Culture Collection, from whom early passage dermal fibroblasts from a normal donor were obtained (ATCC No. CRL 1477). This work was supported by a grant to L.S.L. from the American Heart Association-Louisiana, Inc. C.A.F. is a dissertation fellow of the American Association of University Women and a recipient of the Gerald P. Rodnan Summer Fellowship of the United Scleroderma Foundation. REFERENCES 1. Kishimoto, T., The biology of interleukin-6. Blood 74, l-10, 1989. 2. Le, J., and Vilcek, J., Interleukin-6: A multifunctional cytokine regulating immune reactions and the acute phase protein response. Lab. Invest. 61, 588-602, 1989. 3. Mizel,
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II 12
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P.. and
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of RNA
EFFECTS OF IL-4 ON HUMAN
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36. 37.
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isolation by acid guanidinium thiocyanat+phenol+hloroform extraction. Anal. Biochem. 162, 156-159, 1987. Bonham, L., Lobelle-Rich, P. A., Henderson, L. A., and Levy, L. S., Transforming potential of a myc-containing variant of feline leukemia virus in vitro in early-passage feline cells. J. Virol. 61, 30723081, 1987. Navarro, S., Debili, N., Bernaudin, J.-F., Vainchenker, W., and Doly, J., Regulation of the expression of IL-6 in human monocytes. J. Immunol. 142, 4339-4345, 1989. Shalaby, M. R., Waage, A., and Espevik, T., Cytokine regulation of interleukin 6 production by human endothelial cells. Cell. Immurwl. 121, 372-382, 1989. Walther, Z., May, L. T., and Sehgal, P. B., Transcriptional regulation of the interleukin+,/B cell differentiation factor BSF2/hepatocyte-stimulating factor gene in human fibroblasts by other cytokines. J. Immunol. 140, 974-977, 1988. Elias, J. A., and Lentz, V., IL-l and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA. J. Immunol. 145, 161-166, 1990. Gauchat, J.-F., Gauchat, D., De Week, A. L., and Stadler, B. M., Cytokine mRNA levels in antigen-stimulated peripheral blood mononuclear cells. J. Zmmunol. 19, 1079-1085, 1989. Ray, A., Tatter, S. B., Santhanam, U., Helfgott, D. C., May, L. T., and Sehgal, P. B., Regulation of expression of interleukin6. Ann. N. Y. Acad. Sci. 557, 353-362, 1989. Kohase, M., May, L. T., Tamm, I., Vilcek, J., and Sehgal, P. B., A cytokine network in human diploid fibroblasts: Interactions of 8-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1. Mol. Cell. Biol. 7, 273-280, 1987. Balkwill, F. R., and Burke, F., The cytokine network. Zmmunol. Today 10, 299-304, 1989. Elias, J. A., Freundlich, B., Kern, J. A., and Rosenbloom, J., Cytokine networks in the regulation of inflammation and fibrosis in the lung. Chest 97, 1439-1445,199O. Zhang, Y., Lin, J.-X., Yip, Y. K., and Vilcek, J., Enhancement of CAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: Role in the induction of interleukin 6. Proc. Natl. Acad. Sci. USA 85, 6802-6805, 1988. Van Damme, J., and Van Snick, J., Induction of hybridoma growth factor (HGF), identical to IL-6, in human fibroblasts by
Received November 14, 1991; accepted with revision February 20, 1992
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IMMUNOLOI;~
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IMMUK;oPATHt~LOG\1
Vol. 63, No. 2. May. pp. 182-187. 1992
Human Recombinant Interleukin-4 Induces Proliferation and Interleukin-6 Production by Cultured Human Skin Fibroblasts CAROL A. FEGHALI,* KENNETH *Department
L. BOST,* DENNISW. BOULWARE,~ AND LAURA S.LEVY*-'
and Immunology
of Microbiology
New
and fDepartment of MedLcme, Orleans, Louisiana 70112
The effect of human recombinant interleukin-4 (hrIL-4) on normal human adult dermal fibroblasts in terms of proliferation and IL-6 production was studied. Fibroblasts were exposedto different concentrations of IL-4 for various periods of time. Proliferation was measuredusing a [3H]thymidine incorporation assay. IL-6 production was measured at the transcriptional, protein, and functional levels by Northern blot analysis, radioimmunoassay, and B9 bioassay, respectively. Our results show that hrIL-4 significantly stimulated (two- to fivefold) fibroblasts to increase the incorporation of 13H]thymidine in a dose- and time-dependent manner. However, hrIL-1, hrIL-2, hrIL-5, or hrTNFa, at the sameconcentration (100 U/ml) and for the sametime period (4 days), did not. In addition, IL-4 significantly induced (four- to eightfold) the production of immunoreactive and biologically functional IL-6 However, IL-4 was not as potent an inducer of IL-6 as IL-l. The IL-4-induced IL-6 production was dose and time dependent and was due, at least in part, to a dramatic increase in the steady-state levels of IL-6 mRNA. This is the first report describing the ability of IL-4 to activate human dermal fibroblasts in terms of proliferation and IL-6 production. E mszAcademic PRSS, IW. INTRODUCTION
Interleukin-6 (IL-61 is a cytokine that displays several biological activities. It is produced by a broad range of cells, namely B and T cells, monocytes, keratinocytes, fibroblasts, endothelial cells, and some tumor cells (1). Cell-specific expression of IL-6 results in a multitude of biological responses, including stimulation of B lymphocyte maturation into plasma cells, increase in the production of immunoglobulins, and stimulation of T cell growth and differentiation [reviewed in Refs. (241. In fact, levels of IL-6 have been reported to be elevated during infection, malignancy, autoimmunity, injury, and inflammation [reviewed in Refs.
Tulane
Unlverslty
School
of Meduxne.
fied as a B cell stimulatory factor (91, IL-4 is a lymphokine product of antigen-stimulated helper T cells and has numerous immunoregulatory effects of a stimulatory and inhibitory nature [reviewed in Refs. (3, 10, ll)]. IL-4 exerts its effects on cells that express the IL-4 receptor such as B and T lymphocytes, hematopoietic precursors, fibroblasts, epithelial cells, and endothelial cells (12). Such broad distribution of the IL-4 receptor on cells of various lineages is partly responsible for this cytokine’s diverse effects. The activities of IL-4 include the stimulation of B cell proliferation and differentiation, enhancement of IgG and IgE production, stimulation of thymocyte proliferation, stimulation of T cells (13), and stimulation of bone marrow cell growth (14). IL-4 has also been shown to affect the production of other cytokines (15-18). IL-4 is reported to downregulate IL-6 production by human peripheral blood mononuclear cells (PBMC) (191, but to upregulate IL-6 production in resting human B lymphocytes (201, in keratinocytes (21), and in endothelial cells (221. Little is known about the effect of IL-4 on human fibroblasts, although one report has shown that murine IL-4 can stimulate the proliferation of primary and immortalized murine fibroblasts (23). Another report documents the induction of foreskin fibroblast chemotaxis by human recombinant IL-4 (hrIL-41 (24). We sought to study the effects of IL-4 on human derma1 fibroblasts, particularly with respect to fibroblast proliferation and the production of fibroblast cytokines. We report that exposure to IL-4 stimulates derma1 fibroblasts to proliferate and to produce IL-6. These findings are significant because they suggest a role for IL-4 in activating dermal fibroblasts and in inducing production of IL-6 during inflammation and in certain autoimmune diseases. MATERIALS
AND METHODS
(6-811. IL-4 is also a multifunctional 1 To whom correspondence dressed.
Cells and Culture Reagents
cytokine. First identi-
and reprint
Normal adult dermal fibroblasts from a 22-year-old female were purchased from American Type Culture Collection (ATCC; No. CRL 1477) in passage 4. Cells
requests should be ad182
0090-1229/92
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
EFFECTS OF IL-4 ON HUMAN
were maintained in Dulbecco’s modified Eagle medium (D-MEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml>, streptomycin (100 pg/ml), and amphotericin B (0.25 kg/ml) at 37°C in a humidified atmosphere of 5% COs. Human recombinant IL-18, IL2, IL-4, IL-5, and tumor necrosis factor (Y (TNFa) used in this study were purchased from Amgen Biologicals, Thousand Oaks, California, and had specific activities of 5 X lo8 U/mg, 7 X 10’ U/mg, 2 X 10’ U/mg, 1 X lo6 U/mg, and 2 x lo7 U/mg, respectively. Measurement of Fibroblast Proliferation
Normal adult dermal fibroblasts growing in 25-cm2 tissue culture flasks were trypsinized and deposited in 96-well tissue culture plates at a density of 2000 cells/well. The cells were allowed to adhere for 24 hr. The medium was then replaced by D-MEM supplemented with 10% fetal bovine serum and antibiotic/antimycotic agents. IL-18, IL-2, IL-4, IL-5, or TNFa was added to the test wells, and cells were incubated for 1 to 5 days as described for each experiment. Following a 24-hr pulse with 13Hlthymidine (5 l&i/ml; 20 Ci/mmol, New England Nuclear), the cells were harvested and macromolecular DNA was collected onto Mash II glass fiber filters (Whittaker M.A. Bioproducts). The amount of radiolabel incorporated into DNA was determined by liquid scintillation counting. Measurement of IL-6 Concentration
The levels of immunoreactive Radioimmunoassay. IL-6 in culture supernatants were quantified in duplicate using a radioimmunoassay (Advanced Magnetics, Inc.) as described (25). In this assay, the lower limit of detection of IL-6 is 310 pg/ml.
branes (Nytran, Schleicher & Schuell). The membranes were then hybridized as described (28) to a 32Pradiolabeled probe prepared from the cDNA of human IL-6 (pCSF309, ATCC No. 67153). Following hybridization, membranes were washed as previously described (28) and exposed at -70°C to Kodak XRP-5 film with intensifying screens. Statistical Analysis
The data were analyzed by Student’s t test. A cutoff value of ~0.05 was used to indicate statistical significance. RESULTS
Effect of ZL-4 on Fibroblast Proliferation Effect of dose on proliferation. The proliferation of human dermal fibroblasts cultured in medium supplemented with 0, 30, 100, or 300 U/ml hrIL-4 was measured by 13Hlthymidine incorporation assay. The effect of hrIL-4 on fibroblast proliferation was measured after exposure to varying concentrations for 5 days (Fig. 1). The results show that stimulation of fibroblast proliferation by IL-4 is dose dependent, increasing with increasing IL-4 between 30 and 300 U/ml. The proliferative influence of IL-4 at 100 U/ml was compared to that of other cytokines, specifically IL-l, IL-2, IL-5, or TNFd, when added at the same concentration for the same period of time. The results show that exposure to IL-4 at 100 U/ml for 4 days results in a 2.5-fold stimulation of 13Hlthymidine incorporation, but exposure to other cytokines at the same concentration for the same time period had no stimulatory effect (Fig. 2).
B9 hybridomu proliferation assay. The levels of biologically active IL-6 in culture supernatants were quantified in a specific bioassay using the IL-6dependent murine hybridoma clone B9 (26) as described (25). To confirm the specificity of this assay as reported by others (26), we employed a monoclonal anti-IL-6 antibody (R & D Systems) and observed complete inhibition of B9 cell proliferation in its presence (data not shown). Measurement of IL-6 mRNA Levels
Confluent 25-cm2 tissue culture flasks of normal adult dermal fibroblasts in passage 12 were trypsinized and split 1:3. The medium was removed 24 hr later and replaced by fresh medium supplemented with 100 U/ml IL-l, IL-2, IL-4, or IL-5. As a control, parallel flasks were cultured without the addition of cytokine supplements. Total RNA was prepared from the flasks following the method of Chomczynski and Sacchi (27). The RNA was subjected to electrophoresis in agarose/formaldehyde gels and transferred to nylon mem-
183
FIBROBLASTS
-2 i?
6000
6 L e2
5000
b E -z
3000
5E
1000
z +I 1
4000
2000
C,l-
0
30
100
300
m
hrlL-4 (U/ml) FIG. 1. Proliferation of fibroblasts in response to varying concentrations of hrIL4. Fibroblasts in passage 11 were seeded in 96-well tissue culture plates at a density of 2000 cells/well. Twenty-four hours later, the medium was replaced by 0.2 ml fresh medium supplemented with 0, 30, 100, or 300 U/ml hrIL-4. Following a 4-day incubation, cells were pulsed for 24 hr with [3H1thymidine (1 l&i/well) and then harvested. Macromolecular DNA was collected onto glass fiber filters, and the amount of 13H1thymidine incorporated into DNA was measured by liquid scintillation counting. Open bars represent the mean of 12 independent replicates. Shaded bars represent SEM.
184
FEGHALI
4500 4000 3500 3000 2500 2000 1500 IO00 500
E r,
C
IL-1
IL-2
IL.-4
IL-5
TNFu
I
Culture
conditions
FIG. 2. Proliferation of fibroblasts in response to other cytokines. Fibroblasts in passage 11 were used as in the legend for Fig. 1, except that test media were supplemented with 100 U/ml IL-l, IL-2, IL-4, IL+, or TNFol, and cells were incubated for 3 days before a 24-hr pulse with [3H]thymidine. Shaded bars represent the mean of 12 independent replicates. Black bars represent SEM.
Kinetics
of IL4-Induced
Fibroblast
Proliferation
The kinetics of IL-4-induced proliferation was measured in fibroblasts cultured with 100 U/ml hrIL-4. As a negative control, cells were cultured in parallel in medium without the addition of cytokine. Cells were harvested at 24-hr intervals for 5 days, following a 24-hr pulse with [3Hlthymidine (1 &i/well) before each harvest. As shown in Fig. 3, the results demonstrate that fibroblasts exposed to rIL-4 (100 U/ml) over a period of 5 days respond by significantly increasing the incorporation of [3Hlthymidine relative to control cells (P s 0.05 at Days 3-5). The observed stimulation of fibroblast proliferation is time dependent, gradually increasing over a period of 5 days, whereas proliferation decreases in untreated cells as they reach confluence. Effect of IL4
on IL-6 Production
The production of IL-6 by normal human dermal fibroblasts was measured following exposure to hrIL-4
ET ;\I
or fresh medium supplemented with 100 U/ml of IL-l, IL-2, IL4, or IL-5 for a period of 4 days. Total RNA was then prepared and used in Northern blot analysis. A representative blot, shown in Fig. 6, illustrates that hrIL-4, like hrIL-1, induces significant accumulation of IL-6 mRNA in human dermal fibroblasts (three- to fourfold). IL-4 is not as potent an inducer of IL-6 mRNA as hrIL-1. No alteration in the level of IL-6 mRNA is seen following exposure to hrIL-2 or hrIL-5. These results indicate that the ability of hrIL-4 to induce IL-6 production is due, at least in part, to an accumulation of IL-6 mRNA in human dermal fibroblasts.
1.3 Kb-
FIG. 6. Effect of IL-4 on IL-6 mRNA levels. Fibroblasts were incubated in the presence of 100 U/ml IL-l, IL-2, IL-4, or IL-5 for a period of 4 days. Negative control fibroblasts were maintained in culture medium only, without the addition of any cytokine. Total RNA was prepared and used in Northern blot analysis. Equal amounts of total RNA were loaded in each lane and confirmed by ethidium bromide staining. The experiment was repeated three times.
The essential role played by interleukin-6 in the cytokine network is a subject of intense study. IL-6 serves as an important mediator of the host response to bacterial and viral infection, injury, and malignancy Ireviewed in Refs. (6-8>]. However, like other cytokines, IL-6 does not function in isolation. Rather, it is involved in a complex network of cytokine interactions within which IL-6 exerts its effect on a variety of cells and tissues. The expression of IL-6 is regulated by other cytokines such as interferons, platelet-derived growth factor, epidermal growth factor, IL-l, and IL-2 (6, 10). IL-6 gene expression can be downregulated or upregulated by various cytokines in a cell-type and tissue-specific manner. For example, IL-6 is produced at low levels by unstimulated PBMC (331, and its production is not increased by TNF (34). In contrast, TNF stimulates IL-6 gene expression in fibroblasts (31,35). Further, IL-6 production is downregulated by IL-4 in human PBMC (191, but it is upregulated by the same cytokine in B lymphocytes (201, keratinocytes (21), and endothelial cells (22). Our results show that the secretion of biologically active IL-6 is stimulated in adult human dermal fibroblasts when they are exposed to recombinant IL-4 (Figs. 4 and 5). The stimulation of IL-6 production results, at least in part, from a dramatic accumulation of IL-6 mRNA in dermal fibroblasts exposed to IL-4 (Fig. 6). We report further that IL-4 stimulates the proliferation of human dermal fibroblasts (Figs. 1, 2, and 3). Enhancement of IL-6 production and of fibroblast proliferation by IL-4 is not immediate, and more than 24 hr are necessary for the stimulatory effect to demonstrate statistical significance (P s 0.05). Such a delayed response suggests that an intermediate molecule(s) is produced to mediate the stimulatory effect of IL-4. One possibility for such an intermediate is IL-l, since IL-l is known to be produced by fibroblasts (36), and since it is a potent stimulator of IL-6 (3, 31, 35, 37-39). It is not known whether the production of IL-l by fibroblasts is influenced by IL-4. The role of IL-4 in stimulating IL-6 may be directly relevant in uivo. In scleroderma, an autoimmune connective tissue disorder of the skin and internal organs, mononuclear leukocytes typically infiltrate sites of inflammation. Cellular infiltrates in the skin consist mainly of activated T cells with a helper-inducer phenotype (40, 41). IL-4 is known to be a product of activated T helper cells; thus, IL-4 may be implicated directly in the pathogenesis of scleroderma, particularly as a stimulator of IL-6 production by fibroblasts in affected sites. In fact, we have recently shown that scleroderma fibroblasts produce as much as 30 times more IL-6 than do unaffected fibroblasts from the same patients or from a normal donor (25). Further, scleroderma patients exhibit an elevated ratio of circulating T helper to T suppressor cells (42) as well as increased
186
FEGHALI
T helper activity (43). Recently, IL-4 levels were found to be elevated in supernatants of cultured PBMC from scleroderma patients (441, and detectable IL-4 was reported more frequently in serum from scleroderma patients than from normal donors (45). IL-6 has been implicated in the pathogenesis of other autoimmune disorders as well. IL-6 levels are elevated in the sera of systemic lupus erythematosus patients (461, in the synovial fluid of patients with rheumatoid arthritis (47), in the sera of polymyalgia rheumatica and giant cell arteritis patients (481, and in psoriasis (49). Among the consequences of elevated IL-6 may be the polyclonal B cell activation and the hypergammaglobulinemia observed in inflammatory and autoimmune conditions like scleroderma and others. The data presented here represent the first report of IL-4-induced proliferation, secretion of IL-6, and increase in IL-6 mRNA by human adult dermal fibroblasts. Such stimulatory effects may indeed have a direct role in the pathogenesis of scleroderma, in which the activation of dermal fibroblasts is associated with debilitating fibrosis. IL-4 is further implicated by these data in the pathogenesis of other inflammatory and autoimmune diseases in which an elevation in IL-6 production is observed. ACKNOWLEDGMENTS The authors acknowledge Advanced Magnetics, Inc., who provided radioimmunoassay reagents for the detection of IL-6, and the American Type Culture Collection, from whom early passage dermal fibroblasts from a normal donor were obtained (ATCC No. CRL 1477). This work was supported by a grant to L.S.L. from the American Heart Association-Louisiana, Inc. C.A.F. is a dissertation fellow of the American Association of University Women and a recipient of the Gerald P. Rodnan Summer Fellowship of the United Scleroderma Foundation. REFERENCES 1. Kishimoto, T., The biology of interleukin-6. Blood 74, l-10, 1989. 2. Le, J., and Vilcek, J., Interleukin-6: A multifunctional cytokine regulating immune reactions and the acute phase protein response. Lab. Invest. 61, 588-602, 1989. 3. Mizel,
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Received November 14, 1991; accepted with revision February 20, 1992
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