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• W1993665360 abstract "The role of interleukin-4 as a regulator of immune responses in the skin is investigated with regard to the outcome of contact hypersensitivity reaction in interleukin-4-deficient BALB/C mice. In previous studies conflicting results were obtained concerning the role of interleukin-4 in contact hypersensitivity reactions supporting either a proinflammatory or rather an inhibitory function of this cytokine. Interleukin-4 deficient BALB/C mice sensitized to 2,4-dinitrochlorobenzene showed after challenge a significant reduction in magnitude and duration of the contact hypersensitivity response in comparison with wild-type mice. This attenuation was accompanied by a significant reduction of edema and cellular infiltrates in the dermis and a lacking induction of IL-10 mRNA expression in skin. Also, adoptive transfer experiments revealed that BALB/C mice failed to exhibit contact hypersensitivity after injection of lymph node cells obtained from sensitized interleukin-4 deficient mice. To examine further the role of the contact allergen used to induce the contact hypersensitivity response, mice were also sensitized and challenged with Oxazolone. Here a similar magnitude and duration of contact hypersensitivity in both the interleukin-4 deficient mice and BALB/C control mice was observed. This indicates that the contact hypersensitivity response to 2,4-dinitrochlorobenzene and Oxazolone may partly evolve on different pathways being dependent and independent of interleukin-4. Our results clearly show that the complete loss of endogenous interleukin-4 expression in BALB/C mice is associated with an impaired manifestation of contact hypersensitivity response to 2,4-dinitrochlorobenzene, implying an important proinflammatory function of this cytokine. The role of interleukin-4 as a regulator of immune responses in the skin is investigated with regard to the outcome of contact hypersensitivity reaction in interleukin-4-deficient BALB/C mice. In previous studies conflicting results were obtained concerning the role of interleukin-4 in contact hypersensitivity reactions supporting either a proinflammatory or rather an inhibitory function of this cytokine. Interleukin-4 deficient BALB/C mice sensitized to 2,4-dinitrochlorobenzene showed after challenge a significant reduction in magnitude and duration of the contact hypersensitivity response in comparison with wild-type mice. This attenuation was accompanied by a significant reduction of edema and cellular infiltrates in the dermis and a lacking induction of IL-10 mRNA expression in skin. Also, adoptive transfer experiments revealed that BALB/C mice failed to exhibit contact hypersensitivity after injection of lymph node cells obtained from sensitized interleukin-4 deficient mice. To examine further the role of the contact allergen used to induce the contact hypersensitivity response, mice were also sensitized and challenged with Oxazolone. Here a similar magnitude and duration of contact hypersensitivity in both the interleukin-4 deficient mice and BALB/C control mice was observed. This indicates that the contact hypersensitivity response to 2,4-dinitrochlorobenzene and Oxazolone may partly evolve on different pathways being dependent and independent of interleukin-4. Our results clearly show that the complete loss of endogenous interleukin-4 expression in BALB/C mice is associated with an impaired manifestation of contact hypersensitivity response to 2,4-dinitrochlorobenzene, implying an important proinflammatory function of this cytokine. contact hypersensitivity contact dermatitis 2,4-dinitrobenzoic acid 2,4-dinitrochlorobenzene Despite considerable progress in elucidating the nature of cellular interactions involved in contact hypersensitivity (CHS) reactions neither the role of particular cytokines nor the role of T cell subsets (CD8+ and CD4+ T cells) are fully understood. In vivo and in vitro antibody depletion studies from several laboratories indicated CD4+ T cells as the effector T cells in CHS whereas others observed the ability of CD8+ T cells to mediate CHS (Miller and Jenkins, 1985Miller S.D. Jenkins M.K. In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed hypersensitivity.Cell Immunol. 1985; 92: 414-426Crossref PubMed Scopus (57) Google Scholar;Gosinski and Tigelaar, 1990Gosinski B.L. Tigelaar R.E. Roles of CD4+ and CD8+ T-cells in murine contact hypersensitivity revealed by in vivo monoclonal antibody depletion.J Immunol. 1990; 144: 4121-4128Google Scholar;Hauser, 1990Hauser C. Cultured epidermal Langerhans cells activate effector T-cells for contact sensitivity.J Invest Dermatol. 1990; 95: 436-440Abstract Full Text PDF PubMed Google Scholar;Gautam et al., 1991Gautam S. Matraiano N. Chikkala M. Edinger M. Tubbs R. L3T, 4CD (4+) T-cells that mediate contact hypersensitivity to trinitrochlorobenzene express I-A determinants.Cell Immunol. 1991; 135: 27-41Crossref PubMed Scopus (46) Google Scholar;Xu et al., 1996Xu H. Dilulio N.A. Fairchild R.L. T cell populations primed by hapten sensitisation in contact sensitivity are distinguished by polarised patterns of cytokine production: Interferon (producing (Tc1) effector CD8+ cells and interleukin-4/IL-10 producing (Th2) negative regulatory CD4+cells.J Exp Med. 1996; 183: 1001-1012Crossref PubMed Scopus (373) Google Scholar). The CHS response and delayed-type hypersensitivity reaction are often regarded as Th1-mediated processes, as it could be shown that T-helper 1 cells [interleukin (IL)-2, interferon-γ producers] could passively transfer these reactions (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper T-cell clone. II. Delayed type hypersensitivity is by mediated Th-1 clones.J Immunol. 1987; 138: 3688PubMed Google Scholar;Dieli et al., 1994Dieli F. Asherson G.L. Romano G.C. Sireci G. Gervasi F. Salerno A. IL-4 is essential for the systemic transfer of delayed hypersensitivity by T-cell lines.J Immunol. 1994; 152: 2698-2704PubMed Google Scholar). Studies aimed at defining the role of T-helper 2 cells (IL-4, IL-10 producer) provided evidence that these cells are inactive or may even exert suppressive effects in CHS reactions (Cher and Mosmann, 1987Cher D.J. Mosmann T.R. Two types of murine helper T-cell clone. II. Delayed type hypersensitivity is by mediated Th-1 clones.J Immunol. 1987; 138: 3688PubMed Google Scholar;Asada et al., 1997Asada H. Linton J. Katz S.I. Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4.J Invest Dermatol. 1997; 108: 406-411Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Th2 cells, however, have also been shown to be critically involved in inflammatory processes as, e.g., chronic graft-versus-host reactions, autoimmune diseases and atopic dermatitis (Fanslow et al., 1991Fanslow W.C. Clifford K.N. Park L.S. Rubin A.S. Voice R.F. Beckmann M.P. Widner M.B. Regulation of alloreactivity in vivo by IL-4 and the soluble IL-4 receptor.J Immunol. 1991; 147: 535-542PubMed Google Scholar;Goldman et al., 1991Goldman M. Druet D. Gleichmann E. Th2 cells in systemic autoimmunity: insights from allogeneic diseases and chemically induced autoimmunity.Immunol Today. 1991; 12: 223-228Abstract Full Text PDF PubMed Scopus (373) Google Scholar;Van der Heijden et al., 1991Van der Heijden F.L. Wierenga E.A. Bos J.D. Kapsenberg M.L. High frequency of IL-4 producing CD4+ allergen specific T lymphocytes in atopic dermatitis lesional skin.J Invest Dermatol. 1991; 97: 389-393Abstract Full Text PDF PubMed Google Scholar;Erb et al., 1997Erb K.J. Rüger B. Brevern M. Ryffel B. Schimple A. Rivett K. Constitutive expression of interleukin-4 in vivo causes autoimmune type disorders in mice.J Exp Med. 1997; 185: 329-330Crossref PubMed Scopus (140) Google Scholar). IL-4 is a pleiotropic cytokine product of Th2 cells regulating immunoglobulin isotype switching (IgE, IgG4). Arguably, the most important effect of IL-4 is its role in T cell growth, especially the development of Th2 lineage. It harbors B-cell stimulating activities and can suppress Th1-mediated immune responses (Mosmann and Coffmann, 1989Mosmann T.R. Coffmann R.L. Heterogeneity of cytokine secretion patterns and functions of helper T cells.Adv Immunol. 1989; 46: 111-139Crossref PubMed Scopus (1023) Google Scholar;Mueller et al., 1997Mueller R. Krahl T. Sarvetnick N. Tissue specific expression of interleukin-4 induces extracellular matrix accumulation and extravasation of B-cells.Lab Invest. 1997; 76: 117-128PubMed Google Scholar). It is well known that IL-4 is produced in murine skin during the elicitation phase of CHS (Asada et al., 1997Asada H. Linton J. Katz S.I. Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4.J Invest Dermatol. 1997; 108: 406-411Abstract Full Text PDF PubMed Scopus (78) Google Scholar), but whether it plays a proinflammatory or anti-inflammatory part remains unclear. IL-4 has been reported to exert negative regulatory effects on the CHS response as the injection of recombinant IL-4 reduced the magnitude of the challenge response to the hapten (Gautam et al., 1992Gautam S. Chikkala M. Hamilton T.A. Anti-inflammatory action of IL-4: Negative regulation of contact sensitivity to TNCB.J Immunol. 1992; 148: 1411-1415PubMed Google Scholar). Furthermore, pretreatment with monoclonal anti-IL-4 antibodies before challenge led to increased ear swelling compared with mice pretreated with isotype control monoclonal antibodies (Asada et al., 1997Asada H. Linton J. Katz S.I. Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4.J Invest Dermatol. 1997; 108: 406-411Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Proinflammatory activities of IL-4, however, were suggested from experiments bySalerno et al., 1995Salerno A. Dieli F. Sireci G. Bella via A, Asherson GL. Interleukin-4 is a critical cytokine in contact hypersensitivity.Immunology. 1995; 84: 404-409PubMed Google Scholar using anti-IL-4 antibodies, which markedly inhibited the manifestation of contact dermatitis (CD) and the passive transfer of the CHS reaction by immune lymph node cells. Transgenic mouse technology has enormously advanced the opportunities to study the role of a specific cytokine in CD as the role of genes suspected to be involved in the pathogenesis of CD can now be studied in mice characterized by an overexpression or a loss of function of a particular gene. The IL-4-deficient mice, generated byKühn et al., 1991Kühn R. Rajewsky K. Müller W. Generation and analysis of interleukin-4 deficient mice.Science. 1991; 254: 707-710Crossref PubMed Scopus (1099) Google Scholar, show normal T cell and B cell development, but strongly reduced levels of IgG1 and IgE. Moreover, independently generated IL-4-deficient mice (Kopf et al., 1993Kopf M. Le Gros G. Bachmann M. Lamers M. Bluethmann H. Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine response.Nature. 1993; 362: 245-256Crossref PubMed Scopus (1092) Google Scholar) indicated a requirement of IL-4 in the generation of Th2-derived cytokines and an impairment of immune responses dependent on these cytokines. In order to define better the role of endogenous IL-4 in inducing and regulating CHS we treated BALB/C mice lacking the IL-4 gene with two obligate contact sensitizing agents [2,4-dinitrochlorobenzene (DNCB), Oxazolone] to investigate the CHS response in the absence of IL-4. Breeding pairs of the IL-4-deficient BALB/C mice were a kind gift from Dr. W. Müller, Institute of Genetics, University of Cologne, Germany. They were used in the tenth back-cross generation. Wild-type littermates were purchased from Boongart, Sweden. Mice were bred and kept in specific pathogen free status under standard diet. They were used at 8–16 wk of age. Mice were sensitized topically with 100 μl of a 5% DNCB in an olive oil, acetone solution (1:4) on the ventral unshaved abdomen. Five days later, 10 μl of a 1% DNCB olive oil, acetone solution were each applied to both sides of the right ear. The left ear was exposed to vehicle alone. For CHS to Oxazolone, the same concentrations and vehicle was used for sensitization and challenge. Measurements of ear thickness to determine the cutaneous manifestation of CHS was performed by using a micrometer (Oditest, Kroeplin). The area below the apex of the ear was measured leaving a barely perceptible margin of ear visible above the edge of the measuring device. Ear swelling was calculated by subtracting the prechallenge value from the postchallenge value (Gad et al., 1986Gad S.C. Dunn B.J. Dobbs D.W. Reily C. Walsh R.D. Development and validation of an alternative dermal sensitization test: the mouse ear swelling test.Toxicol Appl Pharmacol. 1986; 84: 93-114Crossref PubMed Scopus (283) Google Scholar). For histologic examination mice were killed 24 h after challenge. Ears were fixed in formalin (10%) for 1–3 d and processed for paraffin embedding. Tissue sections (6 μm) were stained with hematoxylin and eosin, mounted, and analyzed in a blinded fashion by two independent observers. Ears were obtained 12, 24, and 72 h after challenge with DNCB. Ears were snap frozen in liquid nitrogen. Alkaline phosphatase anti-alkaline phosphatase (APAAP) staining of cryosections was performed following established procedures. Rabbit anti-mouse immunoglobulins and APAAP complexes were obtained from Dako (Hamburg, Germany). The alkaline phosphatase reaction was demonstrated by incubation in a solution containing Fast Red TR (1 mg per ml) and naphtol AS-TR phosphate (0.2 mg per ml) (Sigma, St. Louis, MO). Levamisole (0.24 mg per ml) was added to block endogenous alkaline phosphatase activity. Monoclonal anti-mouse antibodies to ICAM-1, and IA (major histocompatibility complex class II) were obtained from Dianova (Hamburg, Germany). Negative controls consisted of the omission of primary antibodies and staining with the dye alone. As a positive control a polyclonal antibody directed against keratin was used. The stained sections were reviewed by two independent observers. T cell proliferation assays were performed in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% antibiotic/antimycotic (Gibco, Karlsruhe, Germany), 10 mM HEPES buffer solution, 1 mM sodium pyruvate, 5 × 10–5 M 2-mercaptoethanol. For fluorescence-activated cell sorting (FACS) analysis, the monoclonal antibody anti-CD4+ (L3T4), anti-CD8+ were used, and anti-rat IgG2aκ (all purchased from PharMingen, San Diego, CA). For magnetic cell sorting isolation a puffer with 2 mM EDTA and 0.5% bovine serum albumin (Sigma) was used. Fluorescence-activated cell sorter analysis was performed to confirm the purity of isolated CD4+ and CD8+ T cells. After magnetic cell sorting T cells were stained with anti-CD4+ (L3T4), anti-CD8+, and anti-rat IgG2aκ as isotype control (all purchased from PharMingen) following the instructions of the manufacturer. CD4+ T cells were 97% and CD8+ T cells were 80% pure (data not shown). Inguinal, axillary, and brachial lymph nodes were obtained 5 d after in vivo sensitization. Single cell suspensions from lymph nodes were purified for CD4+ and CD8+ T cells by magnetic cell sorting as described by the supplier (Milteny Biotec, GmbH Bergisch-Gladbach, Germany). CD4+ T cells were plated at 105 cells per well in the presence of 5 × 105 irradiated (3000 RAD) and 2,4-dinitrobenzoic acid (DNBS)-modified spleen cells as described byShearer, 1974Shearer G.M. Cell-mediated cytotoxicity to trinitrophenyl-modified syngeneic lymphocytes.Eur J Immunol. 1974; 4: 527Crossref PubMed Scopus (436) Google Scholar. T cell lines were restimulated (six to eight times) by adding fresh DNBS-modified irradiated syngeneic stimulator cells every 10–12 d. T cell proliferation was determined by the incorporation of [3H]thymidine the last 17 h of culture. Results are presented as the mean of assays performed in triplicate. Cytokine-specific sandwich enzyme-linked immunosorbent assay for determining quantities of IL-1β, IL-2, IL-4, interferon-γ, and IL-10 in the supernatants from CD4+ and CD8+ cell lines were performed generally following the instructions of the supplier (Genzyme, Cambridge, MA). Cell suspensions obtained from lymph nodes of DNCB-sensitized mice were injected into the ears of naïve mice (five mice per group) (2 × 105 per 20 μl phosphate-buffered saline) as described previously (Dieli et al., 1994Dieli F. Asherson G.L. Romano G.C. Sireci G. Gervasi F. Salerno A. IL-4 is essential for the systemic transfer of delayed hypersensitivity by T-cell lines.J Immunol. 1994; 152: 2698-2704PubMed Google Scholar). The mice were immediately challenged by applying 10 μl 1% DNCB in acetone:olive-oil (4:1) on both sides of the right ear. In these experiments negative controls were groups of mice injected with phosphate-buffered saline, s.c., into the ears and challenged with 1% DNCB as described above. Ear thickness was measured shortly before challenge and swelling was assessed 12 and 24 h after challenge with the use of an engineer’s micrometer (Kroeplin, Schluechtern, Germany) as described. Total RNA was isolated by the guanidium/thiocyanate/caesium chloride method. Reverse transcription was performed according to the manufacturers protocol (Gibco, BRL) using oligo d(T)16 primer. Primers were synthesized by MWG (Ebersberg, Germany) as shown in Table 1.Table 1Primers for RT-PCR were designed by using sequences obtained from GenbankGAPDH 5′: 5′ TCA TGA CCA CAG TCC ATG CCA TCA C 3′GAPDH 3′: 5′ GCC GTA TTA ATT GTC ATA CCA GGA AAT GA 3′IL-10 5′: 5′ ATG CAG GAC TTT AAG GGT TAC TTG GGT T 3′IL-10 3′: 5′ AAT TCG GAG AGA GGT ACA AAC GAG GTT T 3′IL-4 5′: 5′ ATG GGT CTC AAC CAG CTA GT 3′IL-4 3′: 5′ GCT CTT TAG GCT TTC CAG GAA GTC 3′ Open table in a new tab The cycling conditions were as follows: denaturation for 1 min at 94°C, annealing for 1.5 min at 55°C (IL-4, IL-10) and 57°C (GAPDH). Primers, mRNA concentrations, and PCR cycles were titrated to establish standard curves to demonstrate linearity that allowed semiquantitative analysis of signal strength. IL-4 and IL-10 transfected Hy 358 cells were used as positive controls (kindly provided by Dr. W. Müller). After PCR, samples were electrophoresed on agarose gels, blotted and hybridized to P32 end-labeled oligos (IL-4: 5′ AAA ATA TGC GAA GCA CCT TGG AAG CCC TAC 3′; IL-10 5′ GCC TTG TAG ACA CCT TGG TCT TCC AGC TTA 3′) or a P32 labeled random primed GAPDH cDNA probe (kindly provided by Dr. J. Uitto, Thomas Jefferson University, Philadelphia, PA). After hybridization filters were washed and exposed at –80°C to a radiosensitive filter. The double-tailed Student’s t-test was used to compare the significance of differences between groups. Impaired and shortened CHS response to DNCB in the absence of endogenous IL-4To evaluate the capacity of IL-4-deficient mice to develop CHS, 5% DNCB were used for sensitization on the abdomen and 1% DNCB for challenge on the right ear 5 d later. The left ear was exposed to the vehicle (acetone/olive oil) alone. As shown in Figure 1 the time courses of CHS responses were similar in IL-4-deficient mice and control mice. Maximum of ear swelling in both mouse strains was observed after 24 h. Notably, ear swelling responses of IL-4-deficient mice were strongly reduced which exhibited a 40%–60% reduction of ear swelling after 12 h (p < 0.01) compared with control mice. The strongest suppression of CHS in IL-4-deficient mice was observed 12–24 h after challenge with DNCB (Figure 1). After 24 h ear thickness declined in both IL-4-deficient mice and control mice. Mutant mice showed an ear thickness that was back to baseline after 8 d whereas the ear thickness of control mice returned to normal size with a delay of 10 d after challenge. Histologic examination of the challenge and control ears of the wild-type and IL-4-deficient mice after 24 h revealed a reduced edema and cellular infiltrate in the mutant mice confirming the results of the mouse ear swelling test (Figure 2a–d). Furthermore, ICAM-1 and IA (major histocompatibility complex class II) molecule expression as determined by immunohistochemical staining also was markedly reduced after 12 and 24 h in the mutant mice (Figure 2e–j). Differing results were recently reported using IL-4-deficient mice on the genetic background C57BL/6 (Berg et al., 1995Berg D.J. Leach M.W. Kühn R. et al.Interleukin-10 but not interleukin-4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar;Weigmann et al., 1997Weigmann B. Schwing H. Huber H. Ross R. Mossmann H. Knop J. Reske-Kunz A.B. Diminished contact hypersensitivity response in IL-4 deficient mice at a late phase of the elicitation reaction.Scand J Immunol. 1997; 45: 308-314Crossref PubMed Scopus (55) Google Scholar). In order to exclude influences of the genetic background on the diminished CHS response, IL-4-deficient mice were backcrossed to wild-type BALB/C mice in order to generate heterozygous IL-4 (+/–) BALB/C mice. When these heterozygous mice were sensitized and challenged as described above no significant difference in ear swelling response compared with wild-type mice was observed in two experiments performed with five mice per group (data not shown). We analyzed the ability of lymph node cells of sensitized mice to transfer CHS. Cells were injected subcutaneously into the ears of naïve mice followed by challenge with DNCB applied epicutaneously. IL-4-deficient mice were sensitized with 5% DNCB on the abdomen, 5 d later axillary and inguinal lymph nodes were taken and processed to single cell suspensions. Lymph node cells (2 × 105) were injected subcutaneously into the ears of naïve BALB/C mice. The same experiment was performed with lymph node cells of BALB/C mice, which were injected in IL-4-deficient mice as described above. Challenge was performed immediately after injection of lymph node cells by applying 1% DNCB on both sides of the ear. Ear swelling was measured 12 and 24 h after challenge. As expressed in Figure 3 BALB/C mice failed to exhibit CHS after injection of lymph node cells of sensitized IL-4-deficient mice. On the other hand IL-4-deficient mice showed a normal CHS response to DNCB after injection of lymph node cells of sensitized BALB/C mice. To evaluate the CHS reaction to oxazolone, a less potent allergen inducing both a T cell response and immunoglobulin response, we sensitized IL-4-deficient (IL-4 –/–) and BALB/C wild-type mice (IL-4 +/+) with 5% oxazolone. Five days later, mice were challenged with 1% oxazolone and ear swelling was measured as described above. Although mutant mice showed a reduced CHS response in comparison to wild-type BALB/C mice the difference was not significant (Figure 4). These results are in agreement with previous investigations showing a normal CHS reaction in IL-4-deficient C57BL/6 mice to Oxazolone (Berg et al., 1995Berg D.J. Leach M.W. Kühn R. et al.Interleukin-10 but not interleukin-4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar). In order to confirm that IL-4 is adequately induced under the experimental conditions used, semiquantitative reverse transcriptase-PCR for IL-4 was performed. After sensitization with 5% DNCB mice were challenged with 1% DNCB on the right ear. The left ear was exposed to vehicle alone. Twenty-four hours after challenge mice were killed and ears taken for RNA extraction. As shown in Figure 5(a) (lane 2) there is a strong induction of IL-4 mRNA in wild-type mice after 24 h. As expected IL-4 mRNA induction in IL-4-deficient mice (IL-4 –/–) is lacking (Figure 5a, lane 4). IL-10 has previously been shown to be involved in the natural negative control of CHS responses in the skin (Enk et al., 1993Enk A.H. Angeloni V.L. Udey M.C. Katz S.C. Inhibition of DC function by IL-10: a role for the induction of tolerance.J Immunol. 1993; 151: 2390-2396PubMed Google Scholar). Recent studies have shown that after application of an allergen, IL-10 mRNA is rapidly upregulated with a peak at 12 h returning to normal levels thereafter (Ferguson et al., 1994Ferguson T.A. Dube P. Griffith T.S. Regulation of contact hypersensitivity by interleukin-10.J Exp Med. 1994; 179: 1597-1604Crossref PubMed Scopus (152) Google Scholar). Therefore, IL-10 mRNA levels were determined to investigate the role of IL-10 expression in the attenuated CHS response of IL-4-deficient mice. Interestingly, IL-10 mRNA levels were strongly induced after 12 h only in the wild-type mice (Figure 5c, lane 2), whereas no such induction could be detected in the mutant mice (Figure 5c, lane 4) indicating that local up-regulation of IL-10 is not responsible for the reduced CHS response in IL-4-deficient mice. In contrast 12 h after challenge to oxazolone induction of IL-10 mRNA was observed both in control and IL-4-deficient mice (Figure 5e, lanes 2 and 4). In order to evaluate whether an altered proliferation behavior of CD4+ and CD8+ T cells plays a part in the reduced CHS response, T cell proliferation assays were performed. In vivo 5% DNCB-sensitized mice were killed and reactive axillary and inguinal lymph nodes were obtained. Single cell suspensions from these lymph nodes were separated for CD4+ and CD8+ T cells by magnetic cell sorting. Pure CD4+ and CD8+ T cells controlled by fluorescence-activated cell sorter were incubated with syngeneic irradiated and DNBS-modified spleen cells. Proliferation activity was measured by the incorporation of [3H]thymidine the last 17 h of culture. Proliferation indices of CD4+ and CD8+ T cells from IL-4 (+/+) and IL-4 (–/–) mice were similar. CD4+ and CD8+ T cell lines derived from IL-4 (+/+) and IL-4 (–/–) mice were highly specific for the antigen DNBS indicating that IL-4-deficient mice show normal antigen specific T cell proliferation (data not shown). As the results of ear swelling tests showed an attenuation of the CHS response in IL-4-deficient mice and antigen-specific proliferation capacity of T cells was unchanged, hapten-immune CD4+ and CD8+ T cells were stimulated by culture with syngeneic DNBS modified spleen cells. Cytokine profiles produced by CD4+ and CD8+ T cells from in vivo DNCB-sensitized mice were analyzed from the culture supernatants of the cells. IL-4 production was not observed in CD4+ IL-4 (–/–) T cells, as predicted, whereas IL-4 production of control CD4+ T cells reached a maximum 12 h after in vitro restimulation with DNBS. Forty-eight hours after stimulation the IL-4 concentration declined and went to nondetectable levels after 144 h (Figure 6b). IL-2 production of CD4+ T cells from both mouse strains were similar as were IL-1α and IL-1β concentrations (data not shown). Analysis of the cytokine production of CD8+ cells revealed, however, that IL-4-deficient CD8+ T cells produced significantly more IL-10 than cells obtained from control mice (Figure 6a). The duration and magnitude of the CHS response depend on cell–cell interactions, which are generally mediated by a direct contact via receptor–ligand interactions and indirect signaling via cytokines or growth factors. Concerning the role of IL-4 in CHS contradicting results have been obtained (Gautam et al., 1992Gautam S. Chikkala M. Hamilton T.A. Anti-inflammatory action of IL-4: Negative regulation of contact sensitivity to TNCB.J Immunol. 1992; 148: 1411-1415PubMed Google Scholar;Salerno et al., 1995Salerno A. Dieli F. Sireci G. Bella via A, Asherson GL. Interleukin-4 is a critical cytokine in contact hypersensitivity.Immunology. 1995; 84: 404-409PubMed Google Scholar;Asada et al., 1997Asada H. Linton J. Katz S.I. Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4.J Invest Dermatol. 1997; 108: 406-411Abstract Full Text PDF PubMed Scopus (78) Google Scholar) supporting an anti-inflammatory as well as a proinflammatory function of IL-4. WhileGautam et al., 1992Gautam S. Chikkala M. Hamilton T.A. Anti-inflammatory action of IL-4: Negative regulation of contact sensitivity to TNCB.J Immunol. 1992; 148: 1411-1415PubMed Google Scholar demonstrated that injection of recombinant IL-4 protein inhibits the elicitation phase and treatment of mice with anti-IL-4 monoclonal antibody augmented the CHS response,Salerno et al., 1995Salerno A. Dieli F. Sireci G. Bella via A, Asherson GL. Interleukin-4 is a critical cytokine in contact hypersensitivity.Immunology. 1995; 84: 404-409PubMed Google Scholar observed that the treatment of lymph node cells with anti-sense oligonucleotides to IL-4 inhibited the transfer of CHS to recipient mice. Our study aimed to analyze the role of endogenous IL-4 in CHS using transgeneic mice lacking IL-4 expression. Therefore, we induced CHS in BALB/C mice bearing a germline mutation in the IL-4 gene using two different obligate contact allergens: DNCB and oxazolone. CHS was measured quantitatively by using the classic parameter of contact dermatitis, the ear swelling test (Gad et al., 1986Gad S.C. Dunn B.J. Dobbs D.W. Reily C. Walsh R.D. Development and validation of an alternative dermal sensitization test: the mouse ear swelling test.Toxicol Appl Pharmacol. 1986; 84: 93-114Crossref PubMed Scopus (283) Google Scholar). Previously,Berg et al., 1995Berg D.J. Leach M.W. Kühn R. et al.Interleukin-10 but not interleukin-4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar reported a nonsignificant reduction of the CHS to oxazolone in IL-4-deficient mice on the C57BL/6 background, which is known to be a low responder in CHS (Shultz and Baily, 1975Shultz L.D. Baily D.W. Genetic control of contact sensitivity in mice: effect of H-2 and non H-2 loci.Immunogenetics. 1975; 1: 570-583Crossref Scopus (56) Google Scholar).Weigmann et al., 1997Weigmann B. Schwing H. Huber H. Ross R. Mossmann H. Knop J. Reske-Kunz A.B. Diminished contact hypersensitivity response in IL-4 deficient mice at a late phase of the elicitation reaction.Scand J Immunol. 1997; 45: 308-314Crossref PubMed Scopus (55) Google Scholar observed a significant reduction of CHS in the late phase (up to day 3 after challenge) of the CHS response using DNFB as the allergen in the same strain (C57BL/6). BALB/C mice, however, which have a different genetic restriction and are widely used to investigate CHS reactions, were not yet analyzed. This mouse strain is known as a high responder in contact dermatitis (Shultz and Baily, 1975Shultz L.D. Baily D.W. Genetic control of contact sensitivity in mice: effect of H-2 and non H-2 loci.Immunogenetics. 1975; 1: 570-583Crossref Scopus (56) Google Scholar). Using DNCB as the sensitizing allergen, the time course of CHS in BALB/C mutant mice and control mice was characteristic for a CHS response with maximum ear swelling after 24 h. This indicates that contact dermatitis can be induced in mutant BALB/C mice lacking IL-4 expression. Direct comparison of the intensity of the ear swelling response of mutant and control mice, however, revealed that mutant mice showed a significantly attenuated and shortened CHS response from 12 to 194 h after challenge. Histologic examination of the ears revealed that the impaired CHS response in IL-4-deficient mice measured by the mouse ear swelling test was accompanied by decreased cellular infiltrate and edema in the challenged ear. The reduced cellular infiltrate was also accompanied by a diminished induction of ICAM-1 and major histocompatibility complex class II molecules, which could in part be due to the lacking action of IL-4, as this cytokine has previously been shown to induce these molecules (Masinovsky et al., 1990Masinovsky B. Urdal D. Gallatin M. IL-4 acts synergistically with IL-1 to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1.J Immunol. 1990; 145: 2886PubMed Google Scholar;Galea et al., 1993Galea P. Lebranchu Y. Thibault C. Baros P. Interleukin-4 and TNF alpha induce differential adhesion pathways of endothelial cells for the binding of peripheral blood lymphocytes.Scand J Immunol. 1993; 36: 575Crossref Scopus (21) Google Scholar). The attenuated CHS response in the IL-4-deficient mice was restored to normal levels in heterozygous mice excluding an influence of the genetic background. These findings do not support an inhibitory role of IL-4 in the CHS response to DNCB but rather a proinflammatory, as both magnitude and duration of the CHS response were significantly diminished in the IL-4-deficient mice. As our results differ from the observations ofBerg et al., 1995Berg D.J. Leach M.W. Kühn R. et al.Interleukin-10 but not interleukin-4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar the question arose whether the allergens (oxazolone, DNCB) applied or the genetic background is responsible for this discrepancy. Therefore, additional experiments were performed using oxazolone as the sensitizing agent. Notably, as in the C57BL/6 strain, no difference in the ear swelling response compared with wild-type mice was observed. Thus, our results extend the observations ofBerg et al., 1995Berg D.J. Leach M.W. Kühn R. et al.Interleukin-10 but not interleukin-4 is a natural suppressant of cutaneous inflammatory responses.J Exp Med. 1995; 182: 99-108Crossref PubMed Scopus (229) Google Scholar suggesting that the CHS response to oxazolone and DNCB evolves on different pathways independent and dependent on IL-4. Similar results have recently been obtained byShornick et al., 1996Shornick L.P. Togni P. Mariathasan S. et al.Mice deficient in IL-1 (manifest impaired contact hypersensitivity to Trinitrochlorobenzene.J Exp Med. 1996; 183: 1427-1436Crossref PubMed Scopus (218) Google Scholar andZengh et al., 1995Zengh H. Fletcher D. Kozak W. et al.Resistance of fever induction and impaired acute phase response in interleukin-1 beta deficient mice.Immunity. 1995; 3: 9-19Abstract Full Text PDF Scopus (342) Google Scholar. These authors reported that IL-1β-deficient mice exhibited a differential response to trinitrochlorobenzene (TNCB) and oxazolone. A normal CHS response to oxazolone was found, whereas a defective CHS response to low doses of TNCB could be restored using high doses of the sensitizing antigen. Therefore, one may speculate that more than one type of CHS exists or rather different agents induce different immunologic pathways and cytokine cascades. This has already been implicated for irritants (Willis et al., 1993Willis C.M. Stephens C.J.M. Wilkinson J.D. Differential patterns of epidermal leukocyte infiltration in patch test reactions to structurally unrelated chemical irritants.J Invest Dermatol. 1993; 101: 364-370Abstract Full Text PDF PubMed Google Scholar;Willmer et al., 1994Willmer J.L. Burleson F.G. Kayama F. Kanno J. Luster M.I. Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical induced inflammation in mouse skin.J Invest Dermatol. 1994; 102: 915-922Crossref Scopus (194) Google Scholar;Grangsjo et al., 1996Grangsjo A. Leijon-Kuligowski A. Torma H. Roomans G.M. Lindberg M. Different pathways in irritant contact excema? Early differences in the epidermal elemental content and expression of cytokines after application of 2 different irritants.Contact Dermatitis. 1996; 35: 355-360Crossref PubMed Scopus (46) Google Scholar). To investigate whether an impaired antigen-specific proliferation is responsible for the attenuated CHS in IL-4-deficient mice proliferation assays of CD4+ and CD8+ T cells were performed. These experiments revealed that antigen-specific proliferation of CD4+ and CD8+ T cells is unaltered in the absence of endogenous IL-4.Weigmann et al., 1997Weigmann B. Schwing H. Huber H. Ross R. Mossmann H. Knop J. Reske-Kunz A.B. Diminished contact hypersensitivity response in IL-4 deficient mice at a late phase of the elicitation reaction.Scand J Immunol. 1997; 45: 308-314Crossref PubMed Scopus (55) Google Scholar showed that the antigen presentation of Langerhans cells in IL-4-deficient mice is unaltered suggesting that neither an impaired proliferation capacity of T cells nor an impaired antigen presentation from Langerhans cells to T cells is responsible for the reduction of CHS in IL-4-deficient mice. The analysis of the supernatants of antigen-specific CD4+ and CD8+ T cells from IL-4-deficient and control mice revealed a differential pattern of cytokine synthesis. Although IL-2 and IL-1β production was similar in IL-4 +/+ and IL-4 –/– mice, supernatants from CD8+ cells in IL-4-deficient mice showed higher IL-10 concentrations, whereas in mice on a different genetic background (Kopf et al., 1993Kopf M. Le Gros G. Bachmann M. Lamers M. Bluethmann H. Köhler G. Disruption of the murine IL-4 gene blocks Th2 cytokine response.Nature. 1993; 362: 245-256Crossref PubMed Scopus (1092) Google Scholar) a deficiency of IL-10 synthesis had been observed. Interestingly, adoptive transfer experiments revealed that no CHS response could be elicited when lymph node cells from sensitized IL-4-deficient mice were injected into the ears of control mice. This indicates that IL-4 is an important constituent of the cytokine milieu during the early effector phase, as it has been previously suggested by the studies ofSalerno et al., 1995Salerno A. Dieli F. Sireci G. Bella via A, Asherson GL. Interleukin-4 is a critical cytokine in contact hypersensitivity.Immunology. 1995; 84: 404-409PubMed Google Scholar. Further studies are needed, however, e.g., by reconstituting IL-4 in the mutant mice, to examine its exact role further. The reduced ear swelling and inflammatory response after challenge with 1% DNCB is accompanied by a lack of IL-10 mRNA expression in IL-4-deficient mice. This indicates that overproduction of IL-10 in the ear skin, which is thought to be predominantly produced by keratinocytes (Ferguson et al., 1994Ferguson T.A. Dube P. Griffith T.S. Regulation of contact hypersensitivity by interleukin-10.J Exp Med. 1994; 179: 1597-1604Crossref PubMed Scopus (152) Google Scholar), is not responsible for the diminished CHS response. The lacking induction of IL-10 in IL-4-deficient mice rather reflects the largely reduced inflammatory reaction after challenge with DNCB, as IL-10 is normally induced after challenge with oxazolone. Although an induction of other inhibitory cytokines cannot be excluded, our results further provide strong evidence for a direct proinflammatory activity of IL-4 during the early phase of CHS response as proposed by other authors (Müller et al., 1993Müller K.M. Jaunin F. Masouye I. Saurat J.H. Hauser C. Th2 cells mediate IL-4 dependent local tissue inflammation.J Immunol. 1993; 150: 5576-5584PubMed Google Scholar;Trepper, 1994Trepper R. The anti-tumor and proinflammatory actions of IL-4.Res Immunol. 1994; 144: 633-636Crossref Scopus (17) Google Scholar;Salerno et al., 1995Salerno A. Dieli F. Sireci G. Bella via A, Asherson GL. Interleukin-4 is a critical cytokine in contact hypersensitivity.Immunology. 1995; 84: 404-409PubMed Google Scholar). In summary this study shows that, unexpectedly, a loss of endogenously produced IL-4 leads to a significant reduction of the magnitude and duration of the CHS response to DNCB supporting an important proinflammatory function of this cytokine. The normal CHS response to oxazolone in IL-4-deficient mice suggests, however, that this proinflammatory role depends on the chemical compound used to elicit the CHS response. We thank Dr. W. Müller for his generous gift of IL-4-deficient mice. We are indebted to Dr. Thomas Roos and Thomas Hoffmann for reviewing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft Me 939/3-1, Hu 446/2-1 and the “Köln Fortune Programme”. Parts of this work were presented at the XXVIIth meeting of the European Society for Dermatological Research in Rome, Italy October 2–5 1997." @default.
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• W1993665360 title "Inhibition of Allergic Contact Dermatitis to DNCB But Not to Oxazolone in Interleukin-4-Deficient Mice" @default.
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