Effects of Systemic Interleukin-10 Therapy on Psoriatic Skin Lesions: Histologic, Immunohistologic, and Molecular Biology Findings
2001; Elsevier BV; Volume: 116; Issue: 5 Linguagem: Inglês
10.1046/j.0022-202x.2001.01317.x
ISSN1523-1747
AutoresKhusru Asadullah, Markus Friedrich, S. Hanneken, Christoph Rohrbach, Heike Audring, Merle Ebeling, Wolfram Sterry, Athanasios Vergopoulos, Wolf‐Dietrich Döcke, Hans‐Dieter Volk,
Tópico(s)T-cell and B-cell Immunology
ResumoInterleukin-10 is an important anti-inflammatory and immunosuppressive cytokine with major impact on several immune reactions, including regulatory mechanisms in the skin. Recently, we performed a phase II trial in psoriatic patients receiving subcutaneously interleukin-10 over 7 wk. The clinical response suggested that interleukin-10 might represent a novel anti-psoriatic drug. In order to understand better the mode of action and to elucidate the effects of systemic interleukin-10 treatment on the skin immune system, skin punch biopsies from sites different from interleukin-10 injection were analyzed. Biopsies were obtained from the patients before, at the end, and 3 wk after interleukin-10 therapy. The results are reported here. Histologic examination showed a decrease of several parameters reflecting the psoriatic disease activity as acanthosis and extension of the horny layer. Immunohistologic examination demonstrated decreasing numbers of infiltrating T cells, dermal CD1a+ cells, and a diminished proliferation of epidermal cells. Using a novel, quantitative reverse transcriptase–polymerase chain reaction approach a significant shift within the cytokine pattern was found. Interleukin-10 therapy led to a decrease of cutaneous interleukin-8 and interleukin-10 mRNA expression. Whereas no significant changes of interleukin-6, tumor necrosis factor-α, and interferon-γ expression were found, interleukin-4 was strongly upregulated suggesting a shift from a type 1 towards a type 2 cytokine pattern. The changes within the local cytokine pattern seem to be disease-related, as an inverse course was found in a single interleukin-10 nonresponding patient. Our findings demonstrate considerable effects of systemic interleukin-10 application on the skin immune systems, which might contribute to the anti-psoriatic activity of interleukin-10. Interleukin-10 is an important anti-inflammatory and immunosuppressive cytokine with major impact on several immune reactions, including regulatory mechanisms in the skin. Recently, we performed a phase II trial in psoriatic patients receiving subcutaneously interleukin-10 over 7 wk. The clinical response suggested that interleukin-10 might represent a novel anti-psoriatic drug. In order to understand better the mode of action and to elucidate the effects of systemic interleukin-10 treatment on the skin immune system, skin punch biopsies from sites different from interleukin-10 injection were analyzed. Biopsies were obtained from the patients before, at the end, and 3 wk after interleukin-10 therapy. The results are reported here. Histologic examination showed a decrease of several parameters reflecting the psoriatic disease activity as acanthosis and extension of the horny layer. Immunohistologic examination demonstrated decreasing numbers of infiltrating T cells, dermal CD1a+ cells, and a diminished proliferation of epidermal cells. Using a novel, quantitative reverse transcriptase–polymerase chain reaction approach a significant shift within the cytokine pattern was found. Interleukin-10 therapy led to a decrease of cutaneous interleukin-8 and interleukin-10 mRNA expression. Whereas no significant changes of interleukin-6, tumor necrosis factor-α, and interferon-γ expression were found, interleukin-4 was strongly upregulated suggesting a shift from a type 1 towards a type 2 cytokine pattern. The changes within the local cytokine pattern seem to be disease-related, as an inverse course was found in a single interleukin-10 nonresponding patient. Our findings demonstrate considerable effects of systemic interleukin-10 application on the skin immune systems, which might contribute to the anti-psoriatic activity of interleukin-10. antigen-presenting cells Psoriasis Area and Severity Index In 1989, Mosmann and coworkers observed the production of a so-called "cytokine synthesis inhibitory factor" by T helper 2 cell clones (Fiorentino et al., 1989Fiorentino D.F. Bond M.W. Mosmann T.R. Two types of mouse T-helper cell. V. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones.J Exp Med. 1989; 170: 2081-2095Crossref PubMed Scopus (2393) Google Scholar). This factor was later included in the current cytokine nomenclature as interleukin (IL)-10. Today we know that IL-10 is produced by a variety of cells, in particular monocytes, macrophages, B cells, and T cell subsets (De Waal Malefyt et al., 1991aDe Waal Malefyt R. Abrams J. Bennett B. Figdor C.G. de Vries J.E. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.J Exp Med. 1991; 174: 1209-1220Crossref PubMed Scopus (3280) Google Scholar;Spits and De Waal Malefyt, 1992Spits H. De Waal Malefyt R. Functional characterization of human IL-10.Int Arch Allergy Immunol. 1992; 99: 8-15Crossref PubMed Scopus (169) Google Scholar;D'Andrea et al., 1993D'Andrea A. Aste-mezaga M. Valiante N.M. Ma X. Kubin M. Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells.J Exp Med. 1993; 178: 1041-1048Crossref PubMed Scopus (1287) Google Scholar;Asadullah et al., 1999aAsadullah K. Sabat R. Wiese A. Döcke W.D. Volk H.D. Sterry W. Interleukin-10 in cutaneous disorders: Implications for its pathophysiological importance and therapeutical use.Arch Dermatol Res. 1999; 291: 628-636Crossref PubMed Scopus (52) Google Scholar). Numerous in vitro and animal experiments demonstrated the great impact of IL-10 on immunoregulation. It promotes the development of a type 2 cytokine pattern by inhibiting the interferon (IFN) -γ production of T lymphocytes, particularly via the suppression of IL-12 synthesis in accessory cells (D'Andrea et al., 1993D'Andrea A. Aste-mezaga M. Valiante N.M. Ma X. Kubin M. Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells.J Exp Med. 1993; 178: 1041-1048Crossref PubMed Scopus (1287) Google Scholar), as well as the proinflammatory cytokine production and antigen-presenting capacity of antigen-presenting cells (APC) (De Waal Malefyt et al., 1991bDe Waal Malefyt R. Haanen J. Spits H. et al.Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T-cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class major histocompatibility complex expression.J Exp Med. 1991; 174: 915-924Crossref PubMed Scopus (1706) Google Scholar;Fiorentino et al., 1991Fiorentino D.F. Zlotnik A. Mosmann T.R. Howard M. O'Garra A. IL-10 inhibits cytokine production by activated macrophages.J Immunol. 1991; 147: 3815-3822PubMed Google Scholar). Owing to this considerable anti-inflammatory and immunosuppressive capacities, recombinant human IL-10 became a candidate for the therapy of several immune diseases as Crohn's disease (Van Deventer et al., 1997Van Deventer S.J. Elson C.O. Fedorak R.N. Multiple doses of intravenous interleukin-10 in steroid refractory Crohn's disease. Crohn's disease study group.Gastroenterology. 1997; 113: 383-389Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar), transplant rejection (Wissing et al., 1997Wissing K.M. Morelon E. Legendre C. et al.A pilot trial of recombinant human interleukin-10 in kidney transplant recipients receiving OKT3 induction therapy.Transplantation. 1997; 64: 999-1006Crossref PubMed Scopus (48) Google Scholar), and recently psoriasis. In a pilot trial we observed considerable clinical effects in the two out of three psoriatic patients receiving lesional, subcutaneous IL-10 applications (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar). Consequently, we performed an open label phase II trial, including 10 psoriatic patients (Asadullah et al., 1999cAsadullah K. Döcke W.D. Ebeling M. et al.IL-10 treatment of psoriasis—clinical results of a Phase II trial.Arch Dermatol. 1999; 135: 187-192Crossref PubMed Google Scholar). Patients received subcutaneous IL-10 over a 7 wk period. The treatment was well tolerated and systemic anti-psoriatic effects were found in nine of 10 patients. A significant decrease of the psoriasis area and severity index (PASI) from 18.1 to 9.3 was observed. Recently,Reich et al., 1998Reich K. Brück M. Gräfe A. Vente C. Neumann C. Garbe C. Treatment of psoriasis with interleukin-10.J Invest Dermatol. 1998; 6 (letter): 1235-1236Crossref Scopus (55) Google Scholar reported a similar clinical effectiveness (decrease of the PASI score by 67.9%) after 6 wk of IL-10 treatment. These promising data suggest that IL-10 might represent a novel therapeutic approach for the treatment of psoriasis. The mode of action, however, is not fully understood (Asadullah et al., 2000Asadullah K. Döcke W.D. Sabat R. Volk H.D. Sterry W. The treatment of psoriasis with interleukin-10: rationale and review of the first clinical trials.Exp Opin Invest Dr. ugs. 2000; 9: 95-102Crossref PubMed Scopus (38) Google Scholar). In contrast to numerous in vitro and animal investigations, there is only limited information on the immunologic effects of IL-10 therapy in humans. These data are mainly restricted to certain information regarding systemic immunologic alterations (Chernoff et al., 1995Chernoff A.E. Granowitz E.V. Shapiro L. et al.A randomized, controlled trial of IL-10 in humans. Inhibition of inflammatory cytokine production and immune responses.J Immunol. 1995; 154: 5492-5499PubMed Google Scholar;Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar,Asadullah et al., 1999aAsadullah K. Sabat R. Wiese A. Döcke W.D. Volk H.D. Sterry W. Interleukin-10 in cutaneous disorders: Implications for its pathophysiological importance and therapeutical use.Arch Dermatol Res. 1999; 291: 628-636Crossref PubMed Scopus (52) Google Scholar). The systemic immunosuppressive effects we observed (decrease of monocytic HLA-DR expression, IL-12 plasma levels, and responsiveness to recall antigens) as well as a shift towards a type 2 cytokine pattern (increasing proportion of IL-4 producing T cells, increase in IgE serum levels) might be the basis of the anti-psoriatic activity of IL-10 (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar,Asadullah et al., 1999aAsadullah K. Sabat R. Wiese A. Döcke W.D. Volk H.D. Sterry W. Interleukin-10 in cutaneous disorders: Implications for its pathophysiological importance and therapeutical use.Arch Dermatol Res. 1999; 291: 628-636Crossref PubMed Scopus (52) Google Scholar,Asadullah et al., 1999cAsadullah K. Döcke W.D. Ebeling M. et al.IL-10 treatment of psoriasis—clinical results of a Phase II trial.Arch Dermatol. 1999; 135: 187-192Crossref PubMed Google Scholar,Asadullah et al., 2000Asadullah K. Döcke W.D. Sabat R. Volk H.D. Sterry W. The treatment of psoriasis with interleukin-10: rationale and review of the first clinical trials.Exp Opin Invest Dr. ugs. 2000; 9: 95-102Crossref PubMed Scopus (38) Google Scholar). Certainly, however, direct effects of IL-10 on the skin immune system might be crucially involved in the anti-psoriatic mode of action as well. In this study we analyzed the histologic, immunohistologic, and molecular biology effects of systemic IL-10 therapy, demonstrating significant cutaneous immunomodulatory effects. The study was approved by the Institutional Review Board of the Medical Faculty, and written informed consent was obtained from all patients. Serial biopsies were obtained from 10 adult patients with moderate or severe chronic plaque psoriasis undergoing IL-10 therapy. Patients received subcutaneous applications of recombinant human IL-10 (SCH 52000; kindly provided by Essex Pharma, Munich, Germany/Schering Plough Research Institute, Kenilworth, NJ) either 8 µg per kg per d (n = 5) or 20 µg per kg three times per week (n = 5) over a period of 49 d. The treatment was well tolerated and anti-psoriatic effects were found in nine of 10 patients. A significant decrease of the PASI by 55.3 ± 11.5% (mean ± SEM) was observed (p < 0.02) (Asadullah et al., 1999cAsadullah K. Döcke W.D. Ebeling M. et al.IL-10 treatment of psoriasis—clinical results of a Phase II trial.Arch Dermatol. 1999; 135: 187-192Crossref PubMed Google Scholar). Three skin biopsies (4 mm punch biopsies from the center of the same plaque/region) were obtained from each of the 10 patients at each of the following three points in time: 7 d before IL-10 therapy (day - 6), at day 50 (first day after the end of therapy) as well as day 71 (3 wk after the end of therapy). So, altogether 90 biopsies were available for histologic (formaldehyde), immunohistologic (snap frozen), and molecular biology (snap frozen) examination. Hematoxylin–eosin staining and histologic examination of two slices of each skin punch biopsy were performed by an experienced senior scientist as described recently (Asadullah et al., 1999cAsadullah K. Döcke W.D. Ebeling M. et al.IL-10 treatment of psoriasis—clinical results of a Phase II trial.Arch Dermatol. 1999; 135: 187-192Crossref PubMed Google Scholar). The histologic investigations included the evaluation of the main psoriatic parameters such as acanthosis, hyperkeratosis, and parakeratosis, the mitotic activity of the epidermis, papillary edema, dilation and tortuosity of capillaries, and neutrophils in the dermis, the stratum spinosum, as well as in the horny layer, respectively. Cryostat sections (6 µm) were made from the frozen biopsies. Two sections each were stained with antibodies against CD3 (Clone: T3–4B5), CD4 (MT 310), CD8 (DK 25) and CD1a (NA 1/34) (all Dako, Hamburg, Germany), CD54 (ICAM-1, 84H10, Coulter-Immunotech, Krefeld/Hamburg, Germany), and Ki67 (MIB 1, Dianova, Hamburg, Germany), respectively. They were processed using the alkaline phosphatase and monoclonal anti-alkaline phosphatase method as described previously (Kellner et al., 1991Kellner I. Konter U. Sterry W. Overexpression of extracellular matrix receptors (VLA-3, 5 and 6) on psoriatic keratinocytes.Br J Dermatol. 1991; 125: 211-216Crossref PubMed Scopus (26) Google Scholar). Using a 10 × 10 mm2 grating ocular (× 40 magnification) in a light microscope, positive cells were counted in the epidermis and upper dermis. Results (mean value of the two slices) were expressed as cells per mm2. Skin biopsies were removed at the time-points indicated above and immediately snap frozen in guanidinium isothiocyanate solution. Before the analysis and during thawing tissues were homogenized using an Ultraturrax tissue homogenizer (Jahnke and Kunkel, Staufen, Germany). Total RNA was extracted from the biopsies by a matrix method as recommended by the manufacturer (Dianova, Hamburg, Germany). RNA was DNAse-digested and subsequently reverse transcribed as previously described (Murphy et al., 1993Murphy E. Hieny S. Sher A. O'Garra A. Detection of in vivo expression of interleukin-10 using a semi-quantitative polymerase chain reaction method in Schistosoma mansoni infected mice.J Immunol Methods. 1993; 162: 211-223Crossref PubMed Scopus (95) Google Scholar). Therefore, a master solution was prepared by adding 80 µl of 5 × first strand buffer (GibcoBRL, Paisley, U.K.), 40 µl of 100 mM dithiothreitol (GibcoBRL), 40 µl of 10 mM dNTP (Pharmacia Biotech, Uppsala, Sweden), 20 µl of 0.1 mg per ml 5′-pd(T)12-18-3′ (Pharmacia Biotech), 10 µl of 40 U per µl RNasin ribonuclease inhibitor (Promega, Madison, WI), and 20 µl of 1 U per µl RQ1 RNase-free DNAse (Promega). This was sufficient for 10 reactions. Twenty-one microliters of master solution were added to about 0.6–0.8 µg of total RNA and 19 μl of DEPC water. The mixture was incubated at 37°C for 30 min, thereafter for 5 min at 95°C. The DNAse-digested RNA was stored at -20°C. The reverse transcription was started by adding 1 µl of 40 U per µl RNasin ribonuclease inhibitor (Promega) and 1 µl of 200 U per µl MMLV-reverse transcriptase (GibcoBRL) per tube. The mixture was incubated at room temperature for 10 min and thereafter at 37°C for 1 h. The reaction was stopped by incubating at 95°C for 7–10 min. After synthesis cDNA was cooled and stored at -20°C. The expression of each gene transcript in skin biopsies was analyzed by real-time PCR using the ABI PRISM 7700 Sequence Detection System (TaqMan, Perkin-Elmer Biosystems, Weiterstadt, Germany). The method employs the 5′ nuclease activity of Taq polymerase to cleave a nonextendable hybridization probe during the extension phase of the PCR. The approach uses dual-labeled fluorogenic hybridization probes. One fluorescence dye serves as a reporter (FAM, 6-carboxyfluorescein) and its emission spectrum is quenched by the second fluorescent dye (TAMRA, 6-carboxy-tetramethyl-rhodamine). The reactions are monitored in real time during the log phase of product accumulation. The increase of the reporter dye fluorescence intensity during PCR is proportional to the amplification of the target sequence. The cycle number at which the amplification plot crosses a fixed threshold above baseline is defined as threshold cycle (Ct). To control variation in cDNA contents throughout different preparations, all results are related to the concentration of a gene, whose expression remains constant ("housekeeping gene"). Our choice of housekeeping gene consisted in the human hypoxanthine-phosphoribosyl-tranferase gene. To normalize the different amounts of cDNA (ΔCt), we calculated the differences between the mean Ct cytokine and the mean Ct hypoxanthine-phosphoribosyl-tranferase. Relative quantitation was performed using the comparative ΔΔCt method according to the manufacturer's instructions. By subtracting ΔCtbefore therapy from ΔCtafter therapy, ΔΔCt was obtained. The result for the cytokine gene expression after treatment relative to before therapy was given by a unit-less value through the formula 2–ΔΔCt. The PCR reaction was performed in a final volume of 25 µl containing 1 µl cDNA, 12.5 µl Master Mix (TaqMan Universal PCR Master Mix, Perkin Elmer, Applied Biosystems, Weiterstadt, Germany), 1 µl hybridization probe, 6 µl primer mix, and 5.5 µl distilled water. After an initial step of 2 min at 50°C involving activation of uracyl-n-glycosylase and degradation of any pre-existing contaminating RNA sequences, a denaturation and a hot start for AmpliTaq Gold DNA polymerase (Perkin Elmer Biosystems) was performed at 95°C for 10 min. The amplification took place in a two-step PCR (40 cycles; 15 s denaturation step at 95°C and 1 min annealing/extension step at 60°C). The mean Ct values for hypoxanthine-phosphoribosyl-tranferase and the cytokines were calculated from double determinations. Data are presented as mean ± SEM. Statistical analysis was performed using the Wilcoxon test for paired values and the Pearson's coefficient with two-tailed significance levels for correlation analyses. Differences were considered significant at p < 0.05. As the number of patients in the two different dose schedules was small (five patients per group) no statistical comparison between the groups was performed. Histologically, a decrease of epidermal thickness along with a decrease of parakeratosis and the number of mitoses were observed. Figure 1 shows the mean values of these objectively determined parameters. Moreover, the quantity of the infiltrate, including both granulocytes and mononuclear cells as well as the Munro's abscesses decreased, whereas the morphology of the papillary vessels was the same as before. Figure 2 shows typical histologic changes in a single patient. So, overall evaluation of the main psoriatic parameters clearly indicated decreasing disease activity under IL-10 therapy in eight of 10 patients. These data correspond to our clinical findings (Asadullah et al., 1999cAsadullah K. Döcke W.D. Ebeling M. et al.IL-10 treatment of psoriasis—clinical results of a Phase II trial.Arch Dermatol. 1999; 135: 187-192Crossref PubMed Google Scholar).Figure 2Reduction of epidermal thickness, hyperkeratosis, and parakeratosis in a typical patient undergoing IL-10 therapy. Intralesional skin biopsy specimens were obtained from a psoriatic patient before (a), at the end (day 50, b), and 3 wk after IL-10 therapy (day 71, c). A decrease of epidermal thickness along with a decrease of parakeratosis and Munro's abscesses as well as a moderate reduction of the infiltrate is demonstrable. Hematoxylin–eosin staining, Scale bars: 20 µm (a,b) and 50 µm (c), respectively.View Large Image Figure ViewerDownload (PPT) A considerable decrease in the number of CD3+ lymphocytes (infiltrating T cells) and CD1a+ cells (cutaneous APC) was observed. The T cell infiltration showed a clear correlation with the decreasing PASI score (r = 0.72; p < 0.001). Moreover, the number of Ki67-positive epidermal cells declined significantly, indicating a decreasing keratinocyte proliferation. The expression of the adhesion molecule ICAM-1 (CD54) was detectable on basal keratinocytes above the dermal papillae before therapy and cleared during therapy. Moreover, a clear decreasing number of mononuclear CD54+ cells was found in the dermis (Figure 3 and Figure 4). When investigating whether the significant decrease in the number of infiltrating T cells results from an effect on a certain subpopulation, no preferential decrease of either CD4+ or CD8+ cells was found (Table I). Remarkably, in the single patient, who did not respond clinically to IL-10 therapy, no decline in T cell infiltration of lesions was measured and increasing numbers of dermal CD1a+, CD54+ cells were observed. Moreover, the number of proliferating epidermal cells (Ki67+ keratinocytes) increased during the treatment period in this patient (data not shown).Figure 4Immunohistologic changes in a typical patient. Four millimetres punch biopsies were obtained from lesional skin of a patient before (left column), at the end (day 50, middle column), and 3 wk after IL-10 therapy (day 71, right column). Immunohistochemical staining was performed using the alkaline phosphatase and monoclonal anti-alkaline phosphatase technique and antibodies against CD3 (a–c), CD54 (d–f), Ki67 (g–i), and CD1a (j–l). Scale bars: 20 µm.View Large Image Figure ViewerDownload (PPT)Table IImmunohistologic findings during IL-10 therapyaData are mean ± SEM from the 10 patients.No. of positive cells per mm2MarkerBefore therapyAt the end of therapy (day 50)3 wk post- treatment day 71CD3 Epidermis106 ± 15104 ± 1991 ± 17 Dermis381 ± 48242 ± 55bp < 0.05 vs before therapy, Wilcoxon test264 ± 68CD4 Epidermis77 ± 11106 ± 1773 ± 9 Dermis259 ± 48193 ± 32241 ± 40CD8 Epidermis56 ± 1055 ± 1048 ± 8 Dermis148 ± 3091 ± 19105 ± 20a Data are mean ± SEM from the 10 patients.b p < 0.05 vs before therapy, Wilcoxon test Open table in a new tab The course of the cutaneous cytokine mRNA expression is shown in Figure 5. A remarkable shift within the cytokine pattern was observed. The most obvious change was the considerable (5–6-fold) increase in the expression of the Th2 cytokine IL-4. So, a significantly higher IL-4 gene expression level was found at the end of therapy (p = 0.015). Remarkably, the enhanced IL-4 expression persisted even 3 wk after the end of therapy (p = 0.05) (Figure 5). In contrast, the typical Th1 cytokine IFN-γ was almost constantly expressed. This, however, resulted in a long-lasting shift within the immunologic important Th1/Th2 balance (Figure 6). The shift from a Th1 toward a Th2 pattern on the local level corresponds well to the same systemic effect of IL-10 therapy we recently described (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar). Moreover, a significant decrease of cutaneous IL-8 mRNA levels was found.Figure 6IL-10 induced a shift within the cutaneous type 1/type 2 cytokine balance. Intralesional skin biopsy specimens were obtained from 10 psoriatic patients before, at the end (day 50), and 3 wk after IL-10 therapy (day 71). The ratios of the single IFN-γ/IL-4 expression values were calculated. The individual pretreatment levels were considered to be 1. Data are shown as mean ± SEM. *p < 0.05 vs before therapy, Wilcoxon test.View Large Image Figure ViewerDownload (PPT) A decrease in the IL-10 mRNA expression was observed. No significant changes were found with respect to the cutaneous IL-6 and tumor necrosis factor (TNF) -α expression (Figure 5). In order to estimate whether the changes within the cytokine pattern might be associated with the clinical response to therapy, the course of a single nonresponder was compared with that of the other patients who responded well. The nonresponding patient was clinically indistinguishable from the other patients before IL-10 therapy. As in the other patients the biopsies were obtained from always the same site (center of an established plaque). Remarkably, this patient, who showed further progress in disease activity during and in particular after IL-10 therapy, exhibited a different cutaneous cytokine expression pattern compared with that of the other patients. In contrast to the other patients, high IL-10 and low IFN-γ, IL-6, and IL-8 expression was found before therapy (Table II). Moreover, during IL-10 therapy the cytokine pattern was inverse to that of the responders: IL-4 decreased (undetectable at the end of therapy), whereas IL-8 slightly increased (data not shown).Table IIComparison of the pretreatment cytokine expression values between responders vs the single nonresponding patientaData are mean ± SEM of the ΔCt values (× 1000).Responder (n = 9)Nonresponder (n = 1)IFN-γ2.6 ± 0.80.91IL-41.3 ± 0.90.55IL-620.2 ± 7.18.6IL-81200 ± 1004230TNF-α90 ± 1.6100IL-103.6 ± 0.926.5a Data are mean ± SEM of the ΔCt values (× 1000). Open table in a new tab Our investigations demonstrate considerable effects of systemic IL-10 application on the skin immune system, which might contribute to the anti-psoriatic activity of IL-10 therapy. Beside our previous observation that IL-10 therapy suppresses the cutaneous delayed type hypersensitivity response (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar,Asadullah et al., 1999cAsadullah K. Döcke W.D. Ebeling M. et al.IL-10 treatment of psoriasis—clinical results of a Phase II trial.Arch Dermatol. 1999; 135: 187-192Crossref PubMed Google Scholar), this is the first report on this immunomodulatory capacity of IL-10 in humans. 1After typesetting of this paper, a manuscript was published describing similar effects of IL-10, as reported here (Reich et al., 2001Reich K. Garbe C. Blaschke V. Response of psoriasis to interleukin-10 is associated with suppression of cutaneous type 1 inflammation, downregulation of the epidermal interleukin-8/CXCR2 pathway and normalization of keratinocyte maturation.J Invest Dermatol. 2001; 116: 319-332https://doi.org/10.1046/j.1523-1747.2001.01248.xCrossref PubMed Scopus (110) Google Scholar). The histologic (extension of acanthosis and horny layer, frequency of mitosis) and immunohistologic findings (number of infiltrating T cells and proliferating keratinocytes) demonstrated a decrease in parameters typical for the psoriatic disease activity. This supports the clinical observation of beneficial effects of IL-10 therapy in psoriasis. It is unlikely, however, that IL-10 exerts its anti-psoriatic activity via direct effects on keratinocytes. In fact, using corresponding concentrations of recombinant human IL-10 as detectable in plasma during IL-10 therapy, we recently demonstrated an IL-10 nonresponsiveness of activated fresh human keratinocytes as well as HaCaT cells in vitro: neither effects on cytokine formation, surface molecule expression nor on proliferation were observed (Seifert et al., 2000Seifert M. Sterry W. Effenberger E. et al.The antipsoriatic activity of IL-10 is rather caused by effects on peripheral blood cells than by direct effect on human keratinocytes.Arch Dermatol Res. 2000; 292: 164-172Crossref PubMed Scopus (33) Google Scholar). It is likely that IL-10 exerts its anti-psoriatic activity by effects on different immune cell populations present within the skin as well as in the circulation. This in particular includes T cells and APC and their interactions. Psoriasis is regarded as a T cell-dependent (auto)immune disease (Valdimarsson et al., 1986Valdimarsson H. Baker B.S. Johnsdottir I. Fry L. Psoriasis. A disease of abnormal keratinocyte proliferation induced by T lymphocytes.Immunol Today. 1986; 7: 256-259Abstract Full Text PDF PubMed Scopus (273) Google Scholar;Christophers and Sterry, 1993Christophers E. Sterry W. Psoriasis.in: Fitzpatrik T.B. Eisen A.Z. Wolff K. Freedberg I.M. Austen K.F. Dermatology in General Medicine. McGraw-Hill, New York1993: 489-515Google Scholar), probably initiated by presentation of so far unknown "psoriasis-related antigens" by specialized cutaneous APC (Weinstein, 1996Weinstein G.D. Can immunomodulatory molecules work topically for psoriasis?.J Invest Dermatol. 1996; 106: 589Crossref PubMed Scopus (10) Google Scholar;Norris et al., 1997Norris D.A. Traves J.B. Leung D.Y.M. Lymphocyte activation in the pathogenesis of psoriasis.J Invest Dermatol. 1997; 109: 1-4Crossref PubMed Scopus (57) Google Scholar). IL-10 is able to suppress the APC activity of monocytes/macrophages and dendritic cells (De Waal Malefyt et al., 1991aDe Waal Malefyt R. Abrams J. Bennett B. Figdor C.G. de Vries J.E. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.J Exp Med. 1991; 174: 1209-1220Crossref PubMed Scopus (3280) Google Scholar,De Waal Malefyt et al., 1991bDe Waal Malefyt R. Haanen J. Spits H. et al.Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T-cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class major histocompatibility complex expression.J Exp Med. 1991; 174: 915-924Crossref PubMed Scopus (1706) Google Scholar;Fiorentino et al., 1991Fiorentino D.F. Zlotnik A. Mosmann T.R. Howard M. O'Garra A. IL-10 inhibits cytokine production by activated macrophages.J Immunol. 1991; 147: 3815-3822PubMed Google Scholar;Enk and Katz, 1993Enk A.H. Katz S.I. Identification and induction of keratinocyte-derived IL-10.J Immunol. 1993; 149: 92-95Google Scholar;Mitra et al., 1995Mitra R.S. Judge T.A. Nestle F.O. Turka L.A. Nickoloff B.J. Psoriatic skin-derived dendritic cell function is inhibited by exogenous IL-10. Differential modulation of B7-1 (CD80) and B7-2 (CD86) expression.J Immunol. 1995; 154: 2668-2677PubMed Google Scholar). In fact, depressed HLA-DR and CD86 expression as well as TNF-α and IL-12 secretion capacity of circulating peripheral blood monocytes were observed during IL-10 therapy (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar). Although the functional impact of IL-10 treatment on single leukocyte subpopulations present within the skin could not be investigated, here we observed decreasing numbers of APC (CD1a+) and infiltrating T cells (CD3+). This might be both just a secondary consequence of the decreasing disease activity, i.e., an epiphenomenon, or the reflection of a direct IL-10 effect leading to a different behavior of these immune cells (including a distinct homing), crucial for the mode of action. Recently, we found that IL-10 administration influences the peripheral T cell subpopulations with regard to their ex vivo cytokine production (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar). By using a novel real-time reverse transcriptase–PCR approach in order to quantify the mRNA expression of several cytokines simultaneously and more precisely, here we found a remarkable shift within the cutaneous cytokine pattern. IL-10 strongly and long-lastingly induced the cutaneous expression of the type 2 cytokine IL-4. In contrast, no effect on cutaneous IFN-γ expression (type 1) was observed. This resulted in a persistent shift of the immunologic important type 1/type 2 cytokine balance on local level, corresponding well to the systemic effects of IL-10 therapy we described before (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar). According to the predominant expression of IL-2 and IFN-γ and the lack/low-level expression of IL-4 in skin lesions, psoriasis is characterized by a type 1 cytokine pattern (Uyemura et al., 1993Uyemura K. Yamamura M. Fivenson D.F. Modlin R.L. Nickoloff B.J. The cytokine network in lesional and lesion-free psoriatic skin is characterized by a T-helper type 1 cell-mediated response.J Invest Dermatol. 1993; 101: 701-705Abstract Full Text PDF PubMed Google Scholar;Schlaak et al., 1994Schlaak J.F. Buslau M. Jochum W. et al.T cells involved in psoriasis vulgaris belong to the Th 1 subset.J Invest Dermatol. 1994; 102: 145-149Abstract Full Text PDF PubMed Google Scholar). As this immunologic imbalance is considered to be of critical importance in psoriasis (Nickoloff, 1991Nickoloff B.J. The cytokine network in psoriasis.Arch Dermatol. 1991; 127: 871-884Crossref PubMed Scopus (304) Google Scholar;Krueger et al., 1990Krueger J.G. Krane J.F. Carter D.M. Gottlieb A.B. Role of growth factors, cytokines, and their receptors in the pathogenesis of psoriasis.J Invest Dermatol. 1990; 94: 135-140Crossref PubMed Scopus (194) Google Scholar;Asadullah et al., 1999bAsadullah K. Döcke W.D. Volk H.D. Sterry W. Pathophysiological role of cytokines in psoriasis.Dr. ugs Today. 1999; 35: 913-924PubMed Google Scholar), the IL-10-induced shift from a type 1 toward a type 2 pattern on the local level might have contributed to the anti-psoriatic effects of IL-10. This corresponds well to the depressed delayed type hypersensitivity reaction in our patients during therapy, as this reaction is considered to be mediated by type 1 cytokines (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar,Asadullah et al., 2000Asadullah K. Döcke W.D. Sabat R. Volk H.D. Sterry W. The treatment of psoriasis with interleukin-10: rationale and review of the first clinical trials.Exp Opin Invest Dr. ugs. 2000; 9: 95-102Crossref PubMed Scopus (38) Google Scholar). Moreover, similar to that what we found during systemic IL-10 therapy, very recently Walters and coworkers 2Walters I, Ozawa M, Trepicchio W, et al: Narrow band UVB suppresses γ-IFN and IL-12 and increases IL-4 production: in vivo evidence of immune deviation in psoriatic patients. J Invest Dermatol 114:792, 2000 (Abstr.) reported that successful ultraviolet therapy of psoriasis is associated with a considerable increase of cutaneous IL-4 expression. It can be speculated that IL-4 induction is a common phenomenon of different anti-psoriatic therapies and, therefore, it should be considered to use this cytokine itself for the therapy of psoriasis. Finally, the significant depression of the IL-8 mRNA expression might have contributed to the anti-psoriatic activity of IL-10 therapy. IL-8 is a potent proinflammatory cytokine considered to be a crucial mediator in psoriasis. It is overexpressed in untreated psoriasis, acting as a chemoattractant, in particular, for granulocytes cumulating in psoriatic lesions (Gillitzer et al., 1991Gillitzer R. Berger R. Mielke V. Muller C. Wolff K. Stingl G. Upper keratinocytes of psoriatic skin lesions express high levels of NAP-1/IL-8 mRNA in situ.J Invest Dermatol. 1991; 97: 73-79Abstract Full Text PDF PubMed Google Scholar;Lemster et al., 1995Lemster B.H. Carroll P.B. Rilo H.R. Johnson N. Nikaein A. Thomson A.W. IL-8/IL-8 receptor expression in psoriasis and the response to systemic tacrolimus (FK506) therapy.Clin Exp Immunol. 1995; 99: 148-154Crossref PubMed Scopus (86) Google Scholar;Kulke et al., 1996Kulke R. Todt-Pingel I. Rademacher D. Rowert J. Schroder J.M. Christophers E. Co-localized overexpression of GRO-alpha and IL-8 mRNA is restricted to the suprapapillary layers of psoriatic lesions.J Invest Dermatol. 1996; 106: 526-530Crossref PubMed Scopus (58) Google Scholar). A recent report has indicated therapeutic effects of anti-IL-8 antibody therapy in psoriasis. 3Lohner ME, Krueger J, Gottlieb A, et al: Clinical trials of a fully human anti-IL-8 antibody for the treatment of psoriasis. Br J Dermatol 141:989, 1999 (Abstr.) In contrast to evidences from our IL-10 pilot trial (Asadullah et al., 1998Asadullah K. Sterry W. Stephanek K. et al.IL-10 is a key cytokine in psoriasis.J Clin Invest. 1998; 101: 783-794Crossref PubMed Scopus (362) Google Scholar) we found a decrease of the cutaneous IL-10 mRNA expression. This might result from the auto-downregulating capacity of IL-10. No significant effects of systemic IL-10 application on cutaneous TNF-α and IL-6 expression were found. These findings argue against the hypothesis that IL-10 exerts its anti-psoriatic activity by restoring the endogenous IL-10 production or by a broad anti-inflammatory mode, suppressing proinflammatory cytokine expression in general. The stringent correlation of the changes in cytokine expression pattern (increase of IL-4 and decrease of IL-8 expression) and a beneficial clinical and histologic course of psoriasis supports their pathophysiologic relevance. The fact that an inverse course of IL-4 and IL-8 expression was observed in the single patient who did not respond to therapy supports this view. Moreover, a different pretreatment cytokine pattern (no IL-10 deficiency, lower expression of proinflammatory cytokines) indicates that this particular patient might suffer from an immunologically distinct type of psoriasis. Further investigations are necessary to determine whether the individual cutaneous cytokine pattern predicts the response to IL-10 therapy. Our data demonstrate a strong regulatory impact of systemic IL-10 application on the skin immune system. The observed effects offer an explanation for the anti-psoriatic activity of IL-10. Further investigations are necessary to elucidate whether they result from targeting skin immune cells or noncutaneous cells (e.g., circulating immune cells) secondary infiltrating the skin. Moreover, it has to be determined which particular IL-10 effects are crucial for the anti-psoriatic activity. This may lead to even more focused novel therapeutic approaches. This work was partly supported by the Deutsche Forschungsgemeinschaft (Ste 366/7) and Schering-Plough USA/Essex Pharma, Germany.
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