- Email: [email protected]
Increased Interleukin-7 Concentrations in Lesional Skin and in the Sera of Patients with Plaque-Type Psoriasis
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
Vol. 83, No. 1, April, pp. 41–44, 1997 Article No. II964313
Increased Interleukin-7 Concentrations in Lesional Skin and in the Sera of Patients with Plaque-Type Psoriasis1 C. BONIFATI,*,† E. TRENTO,† P. CORDIALI-FEI,† M. CARDUCCI,* A. MUSSI,*,† L. D’AURIA,† F. PIMPINELLI,† M. FAZIO,* AND F. AMEGLIO† *Department of Dermatology and †Laboratory of Clinical Pathology, Institute S. Gallicano, Rome, Italy
(TNF-a, IL-4, GM-CSF, and IFN-g), and increased CD25/IL-2Ra expression on T-lymphocytes (1, 2). Recent data have also helped to clarify the different IL-7-mediated immune-regulatory functions. In particular, IL-7 has been shown to synergize with IL-12 in inducing mature T-lymphocyte proliferation (mainly CD8/ cells), IFN-g production, and stimulation of cytotoxic activity (2). Although IL-7 increases IL-4 (a prototype of TH0/ TH2 cytokine pattern) production in anti-CD3 plus anti-CD28-activated T-lymphocytes, IL-7 preferentially upregulates IFN-g expression in this system both at the mRNA and at the protein level (3). In turn, IFNg induces IL-7 production by keratinocytes (4). Psoriasis is an erythematous–squamous disease in which an abnormal interaction between immunocompetent cells (T-lymphocytes and antigen-presenting cells) and resident skin cells (keratinocytes, fibroblasts, endothelial, and mast cells) induces a massive local production of many different cytokines (IL-1, IL-6, IL8, TNF, GM-CSF, IFN-g), mainly with proinflammatory activities. These molecules sustain both phlogosis and keratinocyte proliferation, representing the principal clinical and histologic aspects of psoriasis (5, 6). From a biochemical point of view, many biological mediators have been extensively studied in psoriasis (6), now considered an example of a disease with a TH1-type immune response (7). To our knowledge, no data on IL-7 concentrations have been reported for this disease, at either the local or the systemic level. Since this cytokine seems to play a key role in driving reciprocal interactions between epithelial cells (including keratinocytes) and T-lymphocytes, the IL-7 levels in extracts of the skin and ‘‘stratum corneum’’ from psoriatic patients were measured. The investigation was also carried out on the sera and supernatants of peripheral blood mononuclear cells (PBMCs) of the same patients. The results obtained have been compared with similar samples from a group of healthy volunteers.
Several cytokines have been shown to be increased in psoriasis, mainly at the local and sometimes at the systemic level. At present, no data concerning the relationships between psoriasis and interleukin-7 (IL-7) are available. This biological modifier regulates immune response by means of its pleomorphic activities, including the ability to stimulate different monocyte functions, such as killing of intracellular pathogens, induction of cytokines, and enhancement of some membrane molecule expression. Study groups consisted of nine psoriatic and nine normal subjects. Using a commercially available immune-enzyme method, IL-7 concentrations were determined in various samples: biopsy and scale exstracts, peripheral blood mononuclear cells (PBMCs) supernatants, and sera. The results show that IL-7 levels are significantly increased both in biopsy and in scale extracts obtained from lesional skin compared to those obtained from nonlesional and normal skin (P at least õ0.01). In addition, the serum values were higher in psoriatic patients than in the controls (P Å 0.003). In contrast, no significant differences were observed in the supernatants of unstimulated PBMCs maintained in culture for 48 hr. These data suggest that IL-7 is involved in some way in the pathomechanisms of psoriasis and that the keratinocyte may be a candidate for psoriatic IL-7 overproduction. q 1997 Academic Press
INTRODUCTION
Interleukin-7 (IL-7) is a single-chain 25-kDa molecule produced by the bone marrow, spleen and thymus stromal/epithelial cells, intestinal epithelial cells, keratinocytes, and monocytes (1). This cytokine was first described as a growth factor for B-cell precursors in vitro. Subsequently, it has been shown that IL-7 exerts pleiotropic effects, such as stimulation of fetal and adult thymocyte growth, promotion of NK/LAK activity, activation of mature T-lymphocytes and monocytes, stimulation of the production of different cytokines
MATERIALS AND METHODS
Nine patients (five males and four females; median age, 47 years; range 31–71) affected with active
1 Work supported in part by ‘‘Progetto Ministero Sanita` 1992: Psoriasi.’’
41
AID
Clin 4313
/
a50d$$$161
02-19-97 11:15:29
0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
clina
AP: Clin
42
BONIFATI ET AL.
plaque-type psoriasis were studied. Disease severity was evaluated by means of the Psoriasis Area and Severity Index (8) (PASI scores: median 16.5; range 6.3– 31.7). At the time of enrollment, none of the patients had received either local or systemic therapy for at least 2 weeks. A complete anamnestic report and clinical and biohumoral check-up revealed no concomitant diseases. Skin Sampling and Extraction Procedures Six-millimeter punch biopsies were taken from the lesional and nonlesional skin (at least 10 cm from the lesion margin) of each patient and from the normal skin of nine healthy volunteers (five males and four females; median age, 46 years; range 34–64). The biopsies were obtained under local anesthesia, using 1% lidocaine without epinephrine, and the subcutaneous fat tissue was dissected by means of a lancet and discarded. Finally, the skin samples were weighed and frozen at 0807C until further processing. No significant differences were found in the weights of the samples from lesional, nonlesional, and normal skin (data not shown). Samples of lesional and nonlesional stratum corneum (namely, psoriatic plaques and uninvolved heels) were also taken from the same patients and from the heels of the control group (nine subjects). The scales were weighed and stored at 0807C until used. The skin biopsies were homogenized in 1 ml of RPMI 1640 by means of a glass homogenizer with a motordriven ground-glass pestle and centrifuged at 10,000 rpm for 15 min at 47C. The extracts were passed through 0.45-mm filters and immediately stored at 0807C. The same extraction procedure was employed for the scales. The weight/volume ratios of the scale samples were adjusted to 100 mg/ml, adding suitable amounts of RPMI 1640. The protein concentrations (see methodology below) determined in these samples provided similar results (data not shown). Blood Collection Ten milliliters of serum and heparin anticoagulated blood was obtained from all the patients and controls. The serum samples were immediately stored at 0807C until used.
(Falcon, Becton–Dickinson, Lincoln Park, NJ) at a concentration of 3 1 106 cells/well/ml in RPMI 1640 medium (Gibco BRL Life Technologies Ltd., Paisley, UK) supplemented with 5% fetal calf serum (FCS, Gibco). After 48 hr, the PBMC cultures were spun at 450g and the supernatants were stored at 0407C until testing. Cytokine Determinations The following commercially available kit was employed for the detection of IL-7: IL-7 EIA (enzyme immunoassay), sensitivity 0.025 pg/ml, 200-ml sample volume. After a previous test, 1:2 dilution in RPMI 1640 both for skin biopsy and for scale extracts was adopted. PBMC supernatants were diluted 1:4. The kit was purchased from R&D Systems Inc. (Minneapolis, MN) and the assays were performed following the manufacturer’s instructions. Both samples and standards were analyzed in duplicate and only variation coefficients of less than 15% were accepted. Protein Measurements The amount of protein in the skin biopsy extracts was measured using the Coomassie protein assay reagent (Pierce Europe), following the manufacturer’s recommendations. Data Normalization The IL-7 determinations related to biopsies or scales were normalized for both weight and protein content, as specified under Results. Due to the similar behavior of the two normalization methods, only one has been presented. When the PBMC supernatants were considered, data were normalized for cell numbers (107 cells). Statistical Analysis Due to the limited number of patients and the unknown data distribution, results were expressed as medians and ranges. Accordingly, the following nonparametric tests were used to compare the differences in the cytokine levels of the different groups: Kruskall– Wallis (KW), Wilcoxon (W), or Mann–Whitney U (MW) tests. Finally, Spearman’s rank correlation test was used to evaluate possible correlations between the variables. RESULTS
Preparation of PBMC Supernatants
IL-7 Levels in Skin Extracts
PBMCs from the patients and the healthy donors were separated by density gradient, according to the standard Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) technique (9). PBMCs were cultured in duplicate for 48 hr at 377C in a CO2 atmosphere in 24-well plates
As shown in Fig. 1, significantly different IL-7 levels were detected in the extracts from lesional, nonlesional, and normal skin when analyzed all together (KW: P Å 0.02). In this case, data normalized for protein content are presented, expressed as picograms per
AID
Clin 4313
/
a50d$$$162
02-19-97 11:15:29
clina
AP: Clin
43
IL-7 IN PSORIASIS
concentrations of PBMC culture supernatants obtained from the two groups were compared. As shown in Fig. 1, no significant differences in the IL-7 amounts were found (psoriasis: median 1.4 pg/107 cells, range 0 – 6.0 vs controls: median 2.0 pg/107 cells, range 0 – 2.42; MW: P Å 0.7). Comparison of Serum IL-7 Levels in Patients and Controls
FIG. 1. IL-7 concentrations in lesional (L), nonlesional (NL), and control (CTR) skin (pg/mg protein) and scale extracts (pg/100 mg of scales) and in PBMC supernatants (SUR PBMC) (pg/107 cells) of plaque-type psoriatic (PSO) and control (CTR) subjects.
milligram of protein. Similar data were observed for weight normalization (not shown). In particular, the IL-7 concentrations of the lesional areas (median 4.3 pg/mg protein, range 1.9–10.7) were statistically higher than those found both in nonlesional psoriatic (median 3.2 pg/mg protein, range 0.6– 4.8; W: P Å 0.01) and in normal skin (median 2.2 pg/mg protein, range 1.5–2.9; MW: P Å 0.01). No significant differences were recorded in the extracts of nonlesional and normal skin (MW: P Å 0.2). IL-7 Levels in Scale Extracts Statistically different IL-7 amounts were observed in comparisons of the concentrations found in the scale extracts of lesional, nonlesional, and normal skin (KW: P Å 0.02, Fig. 1). The data, normalized for 100 mg of scale weight, are shown. Similar data were observed after protein normalization (not shown). A more detailed analysis showed that the IL-7 levels of the lesional stratum corneum (median 2.2 pg/100 mg scales, range 0.8–4.2) were significantly higher than those obtained from the heels of both groups: psoriatic (median 0.6 pg/100 mg scales, range 0.4–0.8; W: P Å 0.03) and normal subjects (median 0.8 pg/100 mg scales, ranges 0.4–1.0). No difference in the IL-7 concentrations was observed between scale extract samples from the heels of the psoriatic and the normal subjects (MW, P Å 0.5). IL-7 Levels in PBMC Supernatants To evaluate whether IL-7 levels spontaneously released by PBMCs of psoriatic patients were higher than those produced by normal individuals, the IL-7
AID
Clin 4313
/
a50d$$$162
02-19-97 11:15:29
The analysis of serum IL-7 evaluated both in patients and in controls showed a significant difference in the cytokine levels of the two groups: patients (median 9.5 pg/ml, range 4.6–28.1) vs controls (median 2.94 pg/ml, range 1.13–5.3; MW: P Å 0.0003). However, no correlation was found between the serum IL-7 levels and the PASI scores calculated in the psoriatic patients (R Å 00.04; P Å 0.9). DISCUSSION
Psoriasis is a frequent, inherited erythematous– squamous disease characterized by a profound immunological deregulation (5, 6). More and more data clearly show increased concentrations of different cytokines in psoriasis at both the local (IL-1, IL2, IL-6, IL8, TGF-a, GM-CSF, TNF-a, IFN-g) and the systemic levels (IL-6, IFN-g, and TGF-b1) (10–16). These molecules are thought to play a pivotal role in the pathogenesis of psoriasis, since they are involved both in the appearance and in the subsequent evolution of psoriatic lesions (5, 6). The production of the above-mentioned modulators is the consequence of the interactions between immunocompetent cells (recruited in the skin from the bloodstream) and resident skin cells, which, in turn, become activated as indicated by increased surface adhesion molecule expression and cytokine production (5). In the present study, our attention was focused on IL-7, a cytokine which has not been studied in this field. In fact, recent reports have stressed its importance in the immunological response and its role as a regulator of the reciprocal interactions between keratinocytes and the immunocompetent cells (1). In particular, IL-7 production is upregulated in IFNg stimulated keratinocytes. In turn, IL-7 (mainly cooperating with other cytokines such as IL-2 and IL-12) stimulates IFN-g production by T-lymphocytes, together with its proliferation (2, 3, 17). Increased IFN-g concentrations have been previously reported in psoriasis at both the skin and the serum levels (10, 14). This finding is relevant since this cytokine is mainly involved in enhancing the antigenpresenting functions of the cells and in inducing the synthesis of various cell adhesion molecules, such as ICAM-1, on the membrane of different cell types (18).
clina
AP: Clin
44
BONIFATI ET AL.
The concomitant T-lymphocyte activation may also be dependent on IL-7, and therefore the subsequent events driving toward the clinical expression and persistence of psoriasis (5) may be IL-7 mediated. To establish whether the psoriatic plaque contained increased IL-7 amounts, we examined the cytokine contents in skin extracts. Our data indicate that IL-7 levels are increased in lesional psoriatic skin compared to those of lesion-free and normal skin, suggesting a greater local production of this molecule. Although the cellular source of IL-7 cannot be established, the keratinocyte may represent one of the candidates for its synthesis (1, 17). This possibility was further confirmed by similar findings observed in scale extracts. In fact, since the stratum corneum mainly represents a keratinocyte population, the data are in agreement with an IL-7 production by these activated cells and subsequent storage in parakeratotic keratinocytes. Since mononuclear cells, such as B-lymphocytes and monocytes, are known to be able to produce IL-7 (1, 17) (increased functions of these cells were previously reported in psoriasis (5)), we studied the spontaneous release of this cytokine in supernatants of PBMCs. As already stated in this paper, no statistical difference between the psoriatic patients and the controls was found. This finding supports the possibility that IL-7 is overproduced at the local level by cells differing from those of circulation. Finally, we measured the IL-7 levels in the sera of both patients and controls. Our results clearly indicate that the IL-7 concentrations were significantly higher in sera of psoriatic patients than in normal serum samples. Serum IL-7 could, therefore, represent the amount of circulating IL-7, overproduced at the lesional skin level. The lack of a significant correlation between the serum IL-7 levels and the PASI scores, evaluated in the same patients, is in partial contrast with the hypothesis raised. However, the PASI score principally represents the extension and not the activity of the disease (8). In conclusion, our results suggest an involvement of IL-7 in psoriasis, although further studies are warranted to elucidate the exact role of this molecule in the cytokine network of psoriatic patients. REFERENCES 1. Costello, R., Imbert, J., and Olive, D., Interleukin-7, a major Tlymphocyte cytokine. Eur. Cytokine Network 4, 253–262, 1993. 2. Priti, T. M., Grant, J. A., and Siegel, J. P., Synergistic effect of IL-7 and IL-12 on human T cell activation. J. Immunol. 154, 5093–5102, 1995.
3. Borger, P., Kauffman, H. F., Postma, D. S., and Vellenga, E., Il7 differentially modulates the expression of IFN-gamma and IL4 in activated human T lymphocytes by transcriptional and posttranscriptional mechanisms. J. Immunol. 156, 1333–1338, 1996. 4. Ariizumi, K., Meng, Y., Bergstresser, P. R., and Takashima, A., IFN-gamma-dependent IL-7 gene regulation in keratinocytes. J. Immunol. 154, 6031–6039, 1995. 5. Baker, B. S., and Fry, L., The immunology of psoriasis. Br. J. Dermatol. 126, 1–9, 1992. 6. Nickoloff, B. J., The cytokine network in psoriasis. Arch. Dermatol. 127, 871–884, 1991. 7. Uyemura, K., Yamamura, M., Fivenson, D. F., Modlin, R. L., and 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. 101, 701–705, 1993. 8. Fredrikson, T., and Patterson, U., Severe psoriasis oral therapy with a new retinoid. Dermatologica 157, 238–244, 1978. 9. Boyum, A., Ficoll–Hypaque method for separating mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. Suppl. 77, 1966. 10. Bonifati, C., Ameglio, F., Carducci, M., Sacerdoti, G., Pietravalle, M., and Fazio, M., Interleukin-1-beta, interleukin-6 and interferon-gamma in suction blister fluids of involved and uninvolved skin and sera of psoriatic patients. Acta Derm. Venereol. (Stockholm) Suppl. 186, 23–24, 1994. 11. Bonifati, C., Carducci, M., Cordiali-Fei, P., Trento, E., Sacerdoti, G., Fazio, M., and Ameglio, F., Correlated increases of tumor necrosis factor-alpha, interleukin-6 and granulocyte monocytecolony stimulating factor levels in suction blister fluids and sera of psoriatic patients: relationships with disease severity. Clin. Exp. Dermatol. 19, 383–387, 1994. 12. Turbitt, M. L., Akhurst, R. J., White, S. I., and McKie, R. M., Localization of elevated transforming growth factor-alpha in psoriatic epidermis. J. Invest. Dermatol. 95, 229–232, 1990. 13. Nickoloff, B. J., Karabin, G. D., Barker, J. N. W. N., Griffiths, C. E. M., Sarma, V., Mitra, R. S., Elder, J. T., Kunkel, S. L., and Dixit, V. M., Cellular localization of interleukin-8 and its inducer tumor necrosis factor in psoriasis. Am. J. Pathol. 138, 129–140, 1991. 14. Gomi, T., Shioara, T., Munakata, T., Imanish, K., and Nagashima, M., Interleukin-1a, tumor necrosis factor-a, and interferon-gamma in psoriasis. Arch. Dermatol. Res. 127, 827–830, 1991. 15. Bonifati, C., Solmone, M., Trento, E., Pietravalle, M., Fazio, M., and Ameglio, F., Serum interleukin-6 as an early marker of therapeutic response to UVB radiation and topical steroids in psoriatic patients. Int. J. Clin. Lab. Res. 24, 112–123, 1994. 16. Bonifati, C., Carducci, M., Mussi, A., Pittarello, A., D’Agosto, G., Fazio, M., and Ameglio, F., The levels of transforming growth factor-b1 are increased in the sera of patients with psoriasis and correlated with disease severity. Eur. J. Dermatol. 6, 486–490, 1996. 17. Sieling, P. A., Sakimura, L., Uyemura, K., Yamamura, M., Oliveros, J., Nickoloff, B. J., Rea, T. H., and Modlin, R. L., Il-7 in the cell-mediated immune response to a human pathogen. J. Immunol. 154, 2775–2783, 1995. 18. Katz, A. M., Rosenthal, D., and Sauder, D. N., Cell adhesion molecules. Structure, function, and implication in a variety of cutaneous and other pathologic conditions. Int. J. Dermatol. 30, 153– 160, 1991.
Received July 12, 1996; accepted with revision November 13, 1996
AID
Clin 4313
/
a50d$$$163
02-19-97 11:15:29
clina
AP: Clin
Vol. 83, No. 1, April, pp. 41–44, 1997 Article No. II964313
Increased Interleukin-7 Concentrations in Lesional Skin and in the Sera of Patients with Plaque-Type Psoriasis1 C. BONIFATI,*,† E. TRENTO,† P. CORDIALI-FEI,† M. CARDUCCI,* A. MUSSI,*,† L. D’AURIA,† F. PIMPINELLI,† M. FAZIO,* AND F. AMEGLIO† *Department of Dermatology and †Laboratory of Clinical Pathology, Institute S. Gallicano, Rome, Italy
(TNF-a, IL-4, GM-CSF, and IFN-g), and increased CD25/IL-2Ra expression on T-lymphocytes (1, 2). Recent data have also helped to clarify the different IL-7-mediated immune-regulatory functions. In particular, IL-7 has been shown to synergize with IL-12 in inducing mature T-lymphocyte proliferation (mainly CD8/ cells), IFN-g production, and stimulation of cytotoxic activity (2). Although IL-7 increases IL-4 (a prototype of TH0/ TH2 cytokine pattern) production in anti-CD3 plus anti-CD28-activated T-lymphocytes, IL-7 preferentially upregulates IFN-g expression in this system both at the mRNA and at the protein level (3). In turn, IFNg induces IL-7 production by keratinocytes (4). Psoriasis is an erythematous–squamous disease in which an abnormal interaction between immunocompetent cells (T-lymphocytes and antigen-presenting cells) and resident skin cells (keratinocytes, fibroblasts, endothelial, and mast cells) induces a massive local production of many different cytokines (IL-1, IL-6, IL8, TNF, GM-CSF, IFN-g), mainly with proinflammatory activities. These molecules sustain both phlogosis and keratinocyte proliferation, representing the principal clinical and histologic aspects of psoriasis (5, 6). From a biochemical point of view, many biological mediators have been extensively studied in psoriasis (6), now considered an example of a disease with a TH1-type immune response (7). To our knowledge, no data on IL-7 concentrations have been reported for this disease, at either the local or the systemic level. Since this cytokine seems to play a key role in driving reciprocal interactions between epithelial cells (including keratinocytes) and T-lymphocytes, the IL-7 levels in extracts of the skin and ‘‘stratum corneum’’ from psoriatic patients were measured. The investigation was also carried out on the sera and supernatants of peripheral blood mononuclear cells (PBMCs) of the same patients. The results obtained have been compared with similar samples from a group of healthy volunteers.
Several cytokines have been shown to be increased in psoriasis, mainly at the local and sometimes at the systemic level. At present, no data concerning the relationships between psoriasis and interleukin-7 (IL-7) are available. This biological modifier regulates immune response by means of its pleomorphic activities, including the ability to stimulate different monocyte functions, such as killing of intracellular pathogens, induction of cytokines, and enhancement of some membrane molecule expression. Study groups consisted of nine psoriatic and nine normal subjects. Using a commercially available immune-enzyme method, IL-7 concentrations were determined in various samples: biopsy and scale exstracts, peripheral blood mononuclear cells (PBMCs) supernatants, and sera. The results show that IL-7 levels are significantly increased both in biopsy and in scale extracts obtained from lesional skin compared to those obtained from nonlesional and normal skin (P at least õ0.01). In addition, the serum values were higher in psoriatic patients than in the controls (P Å 0.003). In contrast, no significant differences were observed in the supernatants of unstimulated PBMCs maintained in culture for 48 hr. These data suggest that IL-7 is involved in some way in the pathomechanisms of psoriasis and that the keratinocyte may be a candidate for psoriatic IL-7 overproduction. q 1997 Academic Press
INTRODUCTION
Interleukin-7 (IL-7) is a single-chain 25-kDa molecule produced by the bone marrow, spleen and thymus stromal/epithelial cells, intestinal epithelial cells, keratinocytes, and monocytes (1). This cytokine was first described as a growth factor for B-cell precursors in vitro. Subsequently, it has been shown that IL-7 exerts pleiotropic effects, such as stimulation of fetal and adult thymocyte growth, promotion of NK/LAK activity, activation of mature T-lymphocytes and monocytes, stimulation of the production of different cytokines
MATERIALS AND METHODS
Nine patients (five males and four females; median age, 47 years; range 31–71) affected with active
1 Work supported in part by ‘‘Progetto Ministero Sanita` 1992: Psoriasi.’’
41
AID
Clin 4313
/
a50d$$$161
02-19-97 11:15:29
0090-1229/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
clina
AP: Clin
42
BONIFATI ET AL.
plaque-type psoriasis were studied. Disease severity was evaluated by means of the Psoriasis Area and Severity Index (8) (PASI scores: median 16.5; range 6.3– 31.7). At the time of enrollment, none of the patients had received either local or systemic therapy for at least 2 weeks. A complete anamnestic report and clinical and biohumoral check-up revealed no concomitant diseases. Skin Sampling and Extraction Procedures Six-millimeter punch biopsies were taken from the lesional and nonlesional skin (at least 10 cm from the lesion margin) of each patient and from the normal skin of nine healthy volunteers (five males and four females; median age, 46 years; range 34–64). The biopsies were obtained under local anesthesia, using 1% lidocaine without epinephrine, and the subcutaneous fat tissue was dissected by means of a lancet and discarded. Finally, the skin samples were weighed and frozen at 0807C until further processing. No significant differences were found in the weights of the samples from lesional, nonlesional, and normal skin (data not shown). Samples of lesional and nonlesional stratum corneum (namely, psoriatic plaques and uninvolved heels) were also taken from the same patients and from the heels of the control group (nine subjects). The scales were weighed and stored at 0807C until used. The skin biopsies were homogenized in 1 ml of RPMI 1640 by means of a glass homogenizer with a motordriven ground-glass pestle and centrifuged at 10,000 rpm for 15 min at 47C. The extracts were passed through 0.45-mm filters and immediately stored at 0807C. The same extraction procedure was employed for the scales. The weight/volume ratios of the scale samples were adjusted to 100 mg/ml, adding suitable amounts of RPMI 1640. The protein concentrations (see methodology below) determined in these samples provided similar results (data not shown). Blood Collection Ten milliliters of serum and heparin anticoagulated blood was obtained from all the patients and controls. The serum samples were immediately stored at 0807C until used.
(Falcon, Becton–Dickinson, Lincoln Park, NJ) at a concentration of 3 1 106 cells/well/ml in RPMI 1640 medium (Gibco BRL Life Technologies Ltd., Paisley, UK) supplemented with 5% fetal calf serum (FCS, Gibco). After 48 hr, the PBMC cultures were spun at 450g and the supernatants were stored at 0407C until testing. Cytokine Determinations The following commercially available kit was employed for the detection of IL-7: IL-7 EIA (enzyme immunoassay), sensitivity 0.025 pg/ml, 200-ml sample volume. After a previous test, 1:2 dilution in RPMI 1640 both for skin biopsy and for scale extracts was adopted. PBMC supernatants were diluted 1:4. The kit was purchased from R&D Systems Inc. (Minneapolis, MN) and the assays were performed following the manufacturer’s instructions. Both samples and standards were analyzed in duplicate and only variation coefficients of less than 15% were accepted. Protein Measurements The amount of protein in the skin biopsy extracts was measured using the Coomassie protein assay reagent (Pierce Europe), following the manufacturer’s recommendations. Data Normalization The IL-7 determinations related to biopsies or scales were normalized for both weight and protein content, as specified under Results. Due to the similar behavior of the two normalization methods, only one has been presented. When the PBMC supernatants were considered, data were normalized for cell numbers (107 cells). Statistical Analysis Due to the limited number of patients and the unknown data distribution, results were expressed as medians and ranges. Accordingly, the following nonparametric tests were used to compare the differences in the cytokine levels of the different groups: Kruskall– Wallis (KW), Wilcoxon (W), or Mann–Whitney U (MW) tests. Finally, Spearman’s rank correlation test was used to evaluate possible correlations between the variables. RESULTS
Preparation of PBMC Supernatants
IL-7 Levels in Skin Extracts
PBMCs from the patients and the healthy donors were separated by density gradient, according to the standard Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) technique (9). PBMCs were cultured in duplicate for 48 hr at 377C in a CO2 atmosphere in 24-well plates
As shown in Fig. 1, significantly different IL-7 levels were detected in the extracts from lesional, nonlesional, and normal skin when analyzed all together (KW: P Å 0.02). In this case, data normalized for protein content are presented, expressed as picograms per
AID
Clin 4313
/
a50d$$$162
02-19-97 11:15:29
clina
AP: Clin
43
IL-7 IN PSORIASIS
concentrations of PBMC culture supernatants obtained from the two groups were compared. As shown in Fig. 1, no significant differences in the IL-7 amounts were found (psoriasis: median 1.4 pg/107 cells, range 0 – 6.0 vs controls: median 2.0 pg/107 cells, range 0 – 2.42; MW: P Å 0.7). Comparison of Serum IL-7 Levels in Patients and Controls
FIG. 1. IL-7 concentrations in lesional (L), nonlesional (NL), and control (CTR) skin (pg/mg protein) and scale extracts (pg/100 mg of scales) and in PBMC supernatants (SUR PBMC) (pg/107 cells) of plaque-type psoriatic (PSO) and control (CTR) subjects.
milligram of protein. Similar data were observed for weight normalization (not shown). In particular, the IL-7 concentrations of the lesional areas (median 4.3 pg/mg protein, range 1.9–10.7) were statistically higher than those found both in nonlesional psoriatic (median 3.2 pg/mg protein, range 0.6– 4.8; W: P Å 0.01) and in normal skin (median 2.2 pg/mg protein, range 1.5–2.9; MW: P Å 0.01). No significant differences were recorded in the extracts of nonlesional and normal skin (MW: P Å 0.2). IL-7 Levels in Scale Extracts Statistically different IL-7 amounts were observed in comparisons of the concentrations found in the scale extracts of lesional, nonlesional, and normal skin (KW: P Å 0.02, Fig. 1). The data, normalized for 100 mg of scale weight, are shown. Similar data were observed after protein normalization (not shown). A more detailed analysis showed that the IL-7 levels of the lesional stratum corneum (median 2.2 pg/100 mg scales, range 0.8–4.2) were significantly higher than those obtained from the heels of both groups: psoriatic (median 0.6 pg/100 mg scales, range 0.4–0.8; W: P Å 0.03) and normal subjects (median 0.8 pg/100 mg scales, ranges 0.4–1.0). No difference in the IL-7 concentrations was observed between scale extract samples from the heels of the psoriatic and the normal subjects (MW, P Å 0.5). IL-7 Levels in PBMC Supernatants To evaluate whether IL-7 levels spontaneously released by PBMCs of psoriatic patients were higher than those produced by normal individuals, the IL-7
AID
Clin 4313
/
a50d$$$162
02-19-97 11:15:29
The analysis of serum IL-7 evaluated both in patients and in controls showed a significant difference in the cytokine levels of the two groups: patients (median 9.5 pg/ml, range 4.6–28.1) vs controls (median 2.94 pg/ml, range 1.13–5.3; MW: P Å 0.0003). However, no correlation was found between the serum IL-7 levels and the PASI scores calculated in the psoriatic patients (R Å 00.04; P Å 0.9). DISCUSSION
Psoriasis is a frequent, inherited erythematous– squamous disease characterized by a profound immunological deregulation (5, 6). More and more data clearly show increased concentrations of different cytokines in psoriasis at both the local (IL-1, IL2, IL-6, IL8, TGF-a, GM-CSF, TNF-a, IFN-g) and the systemic levels (IL-6, IFN-g, and TGF-b1) (10–16). These molecules are thought to play a pivotal role in the pathogenesis of psoriasis, since they are involved both in the appearance and in the subsequent evolution of psoriatic lesions (5, 6). The production of the above-mentioned modulators is the consequence of the interactions between immunocompetent cells (recruited in the skin from the bloodstream) and resident skin cells, which, in turn, become activated as indicated by increased surface adhesion molecule expression and cytokine production (5). In the present study, our attention was focused on IL-7, a cytokine which has not been studied in this field. In fact, recent reports have stressed its importance in the immunological response and its role as a regulator of the reciprocal interactions between keratinocytes and the immunocompetent cells (1). In particular, IL-7 production is upregulated in IFNg stimulated keratinocytes. In turn, IL-7 (mainly cooperating with other cytokines such as IL-2 and IL-12) stimulates IFN-g production by T-lymphocytes, together with its proliferation (2, 3, 17). Increased IFN-g concentrations have been previously reported in psoriasis at both the skin and the serum levels (10, 14). This finding is relevant since this cytokine is mainly involved in enhancing the antigenpresenting functions of the cells and in inducing the synthesis of various cell adhesion molecules, such as ICAM-1, on the membrane of different cell types (18).
clina
AP: Clin
44
BONIFATI ET AL.
The concomitant T-lymphocyte activation may also be dependent on IL-7, and therefore the subsequent events driving toward the clinical expression and persistence of psoriasis (5) may be IL-7 mediated. To establish whether the psoriatic plaque contained increased IL-7 amounts, we examined the cytokine contents in skin extracts. Our data indicate that IL-7 levels are increased in lesional psoriatic skin compared to those of lesion-free and normal skin, suggesting a greater local production of this molecule. Although the cellular source of IL-7 cannot be established, the keratinocyte may represent one of the candidates for its synthesis (1, 17). This possibility was further confirmed by similar findings observed in scale extracts. In fact, since the stratum corneum mainly represents a keratinocyte population, the data are in agreement with an IL-7 production by these activated cells and subsequent storage in parakeratotic keratinocytes. Since mononuclear cells, such as B-lymphocytes and monocytes, are known to be able to produce IL-7 (1, 17) (increased functions of these cells were previously reported in psoriasis (5)), we studied the spontaneous release of this cytokine in supernatants of PBMCs. As already stated in this paper, no statistical difference between the psoriatic patients and the controls was found. This finding supports the possibility that IL-7 is overproduced at the local level by cells differing from those of circulation. Finally, we measured the IL-7 levels in the sera of both patients and controls. Our results clearly indicate that the IL-7 concentrations were significantly higher in sera of psoriatic patients than in normal serum samples. Serum IL-7 could, therefore, represent the amount of circulating IL-7, overproduced at the lesional skin level. The lack of a significant correlation between the serum IL-7 levels and the PASI scores, evaluated in the same patients, is in partial contrast with the hypothesis raised. However, the PASI score principally represents the extension and not the activity of the disease (8). In conclusion, our results suggest an involvement of IL-7 in psoriasis, although further studies are warranted to elucidate the exact role of this molecule in the cytokine network of psoriatic patients. REFERENCES 1. Costello, R., Imbert, J., and Olive, D., Interleukin-7, a major Tlymphocyte cytokine. Eur. Cytokine Network 4, 253–262, 1993. 2. Priti, T. M., Grant, J. A., and Siegel, J. P., Synergistic effect of IL-7 and IL-12 on human T cell activation. J. Immunol. 154, 5093–5102, 1995.
3. Borger, P., Kauffman, H. F., Postma, D. S., and Vellenga, E., Il7 differentially modulates the expression of IFN-gamma and IL4 in activated human T lymphocytes by transcriptional and posttranscriptional mechanisms. J. Immunol. 156, 1333–1338, 1996. 4. Ariizumi, K., Meng, Y., Bergstresser, P. R., and Takashima, A., IFN-gamma-dependent IL-7 gene regulation in keratinocytes. J. Immunol. 154, 6031–6039, 1995. 5. Baker, B. S., and Fry, L., The immunology of psoriasis. Br. J. Dermatol. 126, 1–9, 1992. 6. Nickoloff, B. J., The cytokine network in psoriasis. Arch. Dermatol. 127, 871–884, 1991. 7. Uyemura, K., Yamamura, M., Fivenson, D. F., Modlin, R. L., and 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. 101, 701–705, 1993. 8. Fredrikson, T., and Patterson, U., Severe psoriasis oral therapy with a new retinoid. Dermatologica 157, 238–244, 1978. 9. Boyum, A., Ficoll–Hypaque method for separating mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. Suppl. 77, 1966. 10. Bonifati, C., Ameglio, F., Carducci, M., Sacerdoti, G., Pietravalle, M., and Fazio, M., Interleukin-1-beta, interleukin-6 and interferon-gamma in suction blister fluids of involved and uninvolved skin and sera of psoriatic patients. Acta Derm. Venereol. (Stockholm) Suppl. 186, 23–24, 1994. 11. Bonifati, C., Carducci, M., Cordiali-Fei, P., Trento, E., Sacerdoti, G., Fazio, M., and Ameglio, F., Correlated increases of tumor necrosis factor-alpha, interleukin-6 and granulocyte monocytecolony stimulating factor levels in suction blister fluids and sera of psoriatic patients: relationships with disease severity. Clin. Exp. Dermatol. 19, 383–387, 1994. 12. Turbitt, M. L., Akhurst, R. J., White, S. I., and McKie, R. M., Localization of elevated transforming growth factor-alpha in psoriatic epidermis. J. Invest. Dermatol. 95, 229–232, 1990. 13. Nickoloff, B. J., Karabin, G. D., Barker, J. N. W. N., Griffiths, C. E. M., Sarma, V., Mitra, R. S., Elder, J. T., Kunkel, S. L., and Dixit, V. M., Cellular localization of interleukin-8 and its inducer tumor necrosis factor in psoriasis. Am. J. Pathol. 138, 129–140, 1991. 14. Gomi, T., Shioara, T., Munakata, T., Imanish, K., and Nagashima, M., Interleukin-1a, tumor necrosis factor-a, and interferon-gamma in psoriasis. Arch. Dermatol. Res. 127, 827–830, 1991. 15. Bonifati, C., Solmone, M., Trento, E., Pietravalle, M., Fazio, M., and Ameglio, F., Serum interleukin-6 as an early marker of therapeutic response to UVB radiation and topical steroids in psoriatic patients. Int. J. Clin. Lab. Res. 24, 112–123, 1994. 16. Bonifati, C., Carducci, M., Mussi, A., Pittarello, A., D’Agosto, G., Fazio, M., and Ameglio, F., The levels of transforming growth factor-b1 are increased in the sera of patients with psoriasis and correlated with disease severity. Eur. J. Dermatol. 6, 486–490, 1996. 17. Sieling, P. A., Sakimura, L., Uyemura, K., Yamamura, M., Oliveros, J., Nickoloff, B. J., Rea, T. H., and Modlin, R. L., Il-7 in the cell-mediated immune response to a human pathogen. J. Immunol. 154, 2775–2783, 1995. 18. Katz, A. M., Rosenthal, D., and Sauder, D. N., Cell adhesion molecules. Structure, function, and implication in a variety of cutaneous and other pathologic conditions. Int. J. Dermatol. 30, 153– 160, 1991.
Received July 12, 1996; accepted with revision November 13, 1996
AID
Clin 4313
/
a50d$$$163
02-19-97 11:15:29
clina
AP: Clin