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Psoriasis: comparison of immunological markers in patients with acute and remission phase
JOURNALOF
Dermatological Science Journal of Dermatological Science13 (1996) 118%124
Psoriasis: comparison of immunological markers in patients with acute and remission phase 0. De Piti”, M. Ruffelli”, S. Cadoni”, A. Frezzolini”, G.F. Biavab, R. Simomb, V. Bottari”, G. De Sanctis” “Department ‘Department
of Surgery,
of Dermatology, Division of Immunology, bDepartment of Dermatology,I.D.I.-IRCCS, Clinical Immunology and Allergology ‘Tor
I.D.I.-IRCCS, Rome, Italy Rome, Italy University of Rome, Vergata’,
Rome,
Italy
Received 21 August 1995;revised 7 December 1995;accepted 28 December 1995
Abstract
The immunesysteminvolvement in psoriasishas been documentedby the presenceof activated T-cells both in peripheralblood and in psoriatic skin lesionsand by the intervention of cytokines in the inflammatory process.On this basis,we have undertaken a study in order to examine, in addition to activation markers such as CD25 and CD54 (ICAM-1) on peripheralblood mononuclearcells(PBMNCs) surface,serumlevelsof solubleinterleukin (IL)-2 receptor (sIL-2R), solubleICAM- (sICAM-l), solubleCD4 (sCD4), solubleCD8 (sCD8), p2-microglobulin and fibronectin (FN) in psoriatic patientsanalyzed both in acute and remissionphaseobtainedby topical therapy alone. Our resultsshow that PBMNCs expressingIL-2 receptor (CD25) were increasedboth in percentageand absolute numberin respectto controls, and were not modifiedafter remission.On the contrary, the significantly higher number of CD54+ lymphocytes evaluated in acute psoriasis,showeda reduction during the remissionphase,even if the values persistedhigher than controls. Serum levels of sIL-2R, sICAM-1, sCD4, sCD8 and /YZmicroglobulin were significantly higher than controls both in acute and remissionphase; only FN levels were found to be lower, in patients evaluatedboth in acutepsoriasisand after therapy, in respectto normal donors.On the whole, theseresults seemto indicate the persistenceof both cellular and solubleactivation markerseven in psoriasisremissionphase;in this light, we can supposethat topical therapy alone is not able to efficiently down-regulateactivation mechanisms involved in the pathogenesisof the disease. Keywords:
Psoriasis;Immune system;Solublefactors; Adhesion molecules;Topical therapy
1. Introduction
The relevance of immune and inflammatory factors in the pathogenesis of psoriasis is suggested by the production of cytokines from epi-
dermal keratinocytes even in normal appearing skin, by increased production of inflammatory cytokines from activated T-lymphocytes, and by enhanced functions of dendritic antigen presenting cells [l-8]. In the acute phase, a prevalent
0923-181 l/96/$15.000 1996ElsevierScience IrelandLtd. All rightsreserved PII SO923-1811(96)00517-8
0. De Pith
et al. /Journal
of Dermatological
infiltration of CD4 + and DR + T-lymphocytes is present in the lesional skin, while CD8 + lymphocytes are found in perilesional skin and in remission phase [l]. The presence of soluble immune products such as CD4 and CD8 molecules and of some cytokines in both suction-blister fluid and serum from psoriatic patients accounts for the concomitant involvement of skin-associated and peripheral immune system [9-131. In this context, cytokine-inducible adhesion molecules and some receptor-ligand pairs can regulate lymphocyte traffic in vivo, in relation to both the stage of inflammation and the tissue involvement [14-201. An overexpression of VLA-3, -5, -6 on endothelial cells and the presence of LFA-1 + and ICAM- 1 + infiltrating mononuclear cells have been recently documented in acute psoriasis, while a decrease of the ICAM+ lymphocytes has been shown in the uninvolved skin [21-231. These data strongly support the possibility that circulating lymphocytes could share the same activation markers with skin infiltrating lymphocytes, and our recent work demonstrates a large number of circulating CD54 + lymphocytes in peripheral blood samples from patients with active psoriasis ~241.
In this study we have analyzed, in addition to activation markers on peripheral blood lymphocytes (i.e. CD25 and CD54), serum levels of soluble IL-2 receptor (sIL-2R), soluble ICAM1 (sICAM-l), soluble CD4 (sCD4), soluble CD8 (sCD8), b-microglobulin and fibronectin in patients with acute psoriasis, in order to evaluate the presence of a T-cell activation and homotypic and heterotypic cell-cell adhesion molecule expression. Furthermore, the efficiency of a classical topical treatment on such parameters has been evaluated.
2. Materials and methods 2.1. Patients
Thirty consecutive patients (23 males and 7 females) aging from 20 to 80 years of age, suffering from psoriasis (27 vulgaris and 3 erythrodermic) were studied.
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The PAS1 (psoriasis area and severity index) score ranged from 2 to 44 (median 2 1.3). Furthermore patients were analyzed considering the extension of cutaneous involvement (total body surface area, TBSA) excerpted from PAS1 score and thus subdivided in four groups: TBSA < 25% (I stage): 4 patients 25-50% (II stage): 12 patients 50-75% (III stage): 13 patients < 75% (IV stage): 1 patient All patients were evaluated in acute phase and after two weeks of topical therapy alone, using keratolytic agents and/or steroids. We obtained 22 complete remissions (CR) and 8 partial remissions (PR); the mean reduction of cutaneous involvement in PR was 64 + 13%. Blood samples from 11 healthy donors having a similar sex and age distribution, processed at the same time as those of the patients, were used as controls. 2.2. Peripheral blood lymphocytes phenotypical analysis
Peripheral blood specimens were collected in 10 ml vacutainer tubes (Becton-Dickinson, Mountain View, CA) containing ethylenediaminetetraacetate (EDTA). Complete blood counts and leukocytes differential counts were obtained. Preparation of blood cells for flow cytometry was performed by incubating 0.1 ml of blood for 45 min at 0-4°C with monoclonal antibodies (mAbs). mAbs directed against CD3, CD4, CD8, CD19, CD25, CD56 were obtained from Becton-Dickinson, CDlla and CD54 from Immunotech Int., Marseille, France. The samples were added with fats lysing solution, washed twice with phosphate buffer saline (PBS) and then resuspended in PBS. Flow cytometric analysis was performed with a Becton-Dickinson FACScan equipped with an argon ion laser at 15 mW power and filter sets for fluoresceine and phycoeritrin. For each sample, 10 000 events were evaluated. 2.3. Soluble factors
Enzyme-linked immunosorbent assays (ELISA) test kits, utilizing specific murine mAb-coated
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Table 1 PBL phenotypic analysis in controls (n = 11) and in psoriatic patients (n = 30) evaluated in acute phase and in remission Controls
CD19 CD3 CD4 CD8 CD56
Acute phase
%
a.n.
14*5 71 +8 44k6 27 k 7 15*5
283 + 1385 k 858 + 527 k 293 k
116 492 221 158 120
Remission phase
%
a.n.
10*4 70 * 9 42 f 8 28 +9 14*9
226 + 1583 f 950 f 633 f 317 k
93 384 246 185 138
%
a.n.
11+4 71&7 43 + 8 29 k 7 15+9
225 f 98 1453+315 880 f 221 593 + 154 307 f 122
Data are reported as mean values k S.D. both in percentage (%) and in absolute number (a.n.).
polystirene microwells were employed to detect serum levels of sCD4, sCD8, sIL-2R (T-Cell Diagnostics, Inc. Cambridge, USA), sICAM-1 (R and D Systems, Minneapolis, MN, USA) and p2-microglobulin (Farmos Diagnostica, Finland). Fibronectin was detected by a radial immunodiffusion assay (The Binding Site Ltd., Birmingham). All samples or standards were assayed in duplicate. Intra- and inter-assay variability was less than 3%.
number (479 &- 253 mm3 vs. 166 f 102 mm3; P < 0.001) were significantly increased, when compared with normal donors. After therapy, CD54 + circulating lymphocytes decreased in both percentage (15.2 k 6.4%; P < 0.001) and absolute number (320 ) 170 mm3; P < O.OOl), even if they never returned to normal values (Fig. 2).
2.4. Statistical analysis
Serum levels of sIL-2R, during the acute phase, were significantly higher than controls (863 +419 U/ml vs. 584 f 140 U/ml; P <0.02), and were not modified after remission (723 + 506 U/ml) (Fig. 3).
Analysis of significance involved paired and unpaired Student’s two-tailed t-test, and P values less than 0.05 were considered significant.
3.2. Serum levels of siL-2R
3.3. Serum levels of sICAM-1 3. Results 3.1. Phenotypical analysis of PBMCs
In acute psoriasis patients, we found normal levels of CD19+, CD3+, CD4+, CD8+ and CD56+ circulating lymphocytes, which were not modified in the remission phase (Table 1). PBMCs expressing IL-2 receptor (CD25) were increased both in percentage (8.5 + 5.5% vs. 2.7 f 0.8%; P < 0.001) and in absolute number (188 f 145 mm3 vs. 52 + 13 mm3; P
Serum levels of sICAM-1 evaluated in acute phase were significantly higher than normal donors (448 f 170 rig/ml vs. 222 f 63 rig/ml; P < 0.001). After therapy we observed a significant decrease (353 + 92 rig/ml; P < 0.02), even if these values remained above control mean values (P < 0.001) (Fig. 4). 3.4. Serum levels of sCD4 and sCD8
Large amounts of soluble molecules released from activated T-cells, in comparison with normal donors, were also found in sera from patients with acute psoriasis. Levels of both sCD4 (71 + 40 U/ml vs. 29 & 10 U/ml; P < 0.001) and
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* *
20 18 --
*
*
16 -$b 14 -3 12 -I
Fig. 1. Absolute number and percentage of CD25 + PBL in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
sCD8 (521 + 271 U/ml vs. 279 f 69 U/ml; P < 0.001) were higher than controls and were not reduced after the remission, even if sCD8 showed a trend toward a reduction (418 + 278 U/ml; P = 0.07) (Figs. 5 and 6). 3.5. /32-microglobulin
/32-microglobulin levels, higher than controls (2.9 +_ 1.1 pg/ml vs. 1.3 + 0.3 pg/ml; P < O.OOl), significantly decreased in the remission (2.4 f 0.7; P~0.05) (Fig. 7). 3.6. Fibronectin
Fibronectin was found to be reduced in both acute phase (339 + 112 mg/l vs. 529 + 163 mg/ml; P < 0.001) and remission (320 f 97; P < 0.001) (Fig. 8).
4. Discussion Our results demonstrate that some cellular and soluble activation markers are present in bloodstream of psoriatic patients independent of the disease activity. The rise of cell membrane-associated and soluble form of CD25, as well as the similar feature
observed for sCD4 and sCD8 molecules, suggests a chronic T-cell activation. To this regard, literature data report that T-cell activation may be driven by some proinflammatory cytokines which have been found both in the lesional skin [6,7,9,10,25] and in peripheral blood of psoriatic patients [12] in acute as well as in remission phase. In our view it is likely that such activation markers may result from a prevalent skin immune system involvement and that topical reducent therapy alone is able to improve the cutaneous inflammatory process, but not to modulate the underlying immune mechanism. On the contrary, it has been reported that prolonged systemic treatments (i.e. corticosteroids or cyclosporine) are efficient to down-regulate T-cell activation as demonstrated by the reduction of sIL-2R serum levels [26]. Similarly, the high serum levels of /?2-microglobulin, the invariant chain of MHC-I molecules, reflect the rate of activation and proliferation of cutaneous cells (i.e. keratinocytes, Langerhans, etc.); its persistence at high levels after local therapy further supports the presence of a residual activation of the skin immune system. ICAMoverexpression seems to be related to the acute inflammatory process, as demonstrated by a positive correlation between the number of circulating CD54+ lymphocytes and the extension of the cutaneous involvement [24], and
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CD54
Fig. 2. Absolute number and percentage of CD54+ PBL in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001); 0 indicates statistical significance calculated by paired Student’s ‘-test between acute and remission phase (P i 0.05).
the persistence of abnormal numbers of CD54 + circulating lymphocytes in remission phase confirms that cutaneous immunoflogosis is still present. Furthermore, the presence of high levels of sICAM-I even during the remission phase suggests that the rise of adhesion molecules may be related to an inadequate inhibition of up-regulatory inflammatory cytokine production by local therapy alone; also it has been demonstrated that a residual cytokine production is present in psori-
Fig. 3. Serum levels of soluble IL-2 receptor in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Students t-test versus controls (P < 0.02).
atic patients treated with systemic therapy [27]. The activation of adhesive processes could be at least in part responsible for the significantly lower fibronectin serum levels detected in our patients, contrary to recent literature data that report high levels of fibronectin in plasma of psoriatic patients [28]. In our opinion, soluble fibronectin could be recruited into lesional skin by cutaneous cell over-
Fig. 4. Serum levels of soluble ICAM- in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P
0. De Pith
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PfW 4 395 3
23 2 13
RP.
R.P.
Fig. 5. Serum levels of soluble CD4 in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.) * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
Fig. 7. Serum levels of soluble p2-microglobulin in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001); indicates statistical significance calculated by paired Student’s t-test between acute and remission phase (P < 0.05).
expressing specific receptors, or by circulating lymphocytes and polymorphonuclears which actively carry fibronectin molecules on their cell membrane, subtracting them from peripheral bloodstream. In this regard Fyrand demonstrated the presence of large amounts of fibronectin into lesional skin of patients affected by psoriasis vulgaris [29].
In conclusion, the presence of both cellular and soluble activation markers in the bloodstream during acute psoriasis and remission phases seems to account for a systemic involvement of the disease. However, we can also suppose that topical therapy alone is able to induce only its cosmetic effect, irrespective of an efficient immunomodulation. ) Fibrooectin
Iti 700 T 6ol’ 500 400
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Fig. 6. Serum levels of soluble CD8 in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
0 i
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* * k!L A.P. UP.
Fig. 8. Serum levels of soluble fibronectin in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
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References [l] Jablonska S, Glinski W: Overview of Immunology. In: Psoriasis. Edited by HH Roenigk Jr, HI Maibach. Marcel Dekker, New York, 1991, pp. 261-283. [2] Term T, Aiba S, Kato T, Tanaka T, Tagemi H: HLA-DR antigen expression on keratinocytes in higly inflamed parts of psoriatic lesions. Br J Dermatol 116: 87-93, 1987. [3] Phillips TJ, Dover JS: Recent advances in dermatology. N Engl J Med 326: 167-176, 1992. [4] Baadsgard 0, Fisher G, Voorhees JJ, Cooper KD: The role of immune system in the pathogenesis of psoriasis. J Invest Dermatol 95: 325-345, 1990. [5] Krueger JG, Gottlieb AB: Fattori di crescita, citochine ed eicosanoidi. In: Psoriasi. Edited by ISED. Brescia, 1993, pp. 18-28. [6] Krueger JG, Krane JF, Carter DM, Gottlieb AB: Role of growth factors, cytokines and their receptors in the pathogenesis of psoriasis. J Invest Dermatol 94(Suppl 6): 1355-1405, 1990. [7] Kupper TS. Role of epidermal cytokines. In: Immunophysiology. Edited by JJ Oppenheim, EM Shevach. Oxford University Press, 1990, pp. 285-305. [8] Jones JL, Berth-Jones J, Fletcher A, Hutchinson PE: Assessment of epidennal dendritic cell markers and TLymphocyts in psoriasis. J Path01 174: 77-82, 1994. [9] Grossman RM, Gottlieb AB, Seghal PB, Krueger J, May LT, Kupper T, Murphy DP, Ray A: Increased IL-6 in psoriatic plaques. J Invest Dermatol 92: 437, 1989. [lo] Scheynius A, Fransson J, Johansson C, Hammar H, Baker B, Fry L, Valdimarsson H: Expression of interferon-gamma receptors in normal and psoriatic skin. J Invest Dermatol 98(2): 255-258, 1992. [ll] Vollmer S, Menssen A, Trommler P, Schendel D, Prim JC: T lymphocytes derived from skin lesions of patients with psoriasis vulgaris express a novel cytokine pattern that is distinct from that of T helper type 1 and T helper type 2 cells. Eur J Immunol 24: 237772382, 1994. [12] Gomi T, Shiohara T, Munakata T, Imanishi K, Nagashima M: Interleukin I-alfa, tumor necrosis factor alfa, and interferon gamma in psoriasis. Arch Dermatol 127: 827-830, 1991. [13] Takematsu H, Tagami H: Determination of released CD4 and CD8 antigens in the suction-blister fluid and hornytissue extract in patients with psoriasis. Br J Dermatol 124: 550-554, 1991. [14] Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA: Induction by IL-1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol 137: 18931896, 1986. [15] Griffiths CEM, Voorhees JJ, Nickoloff BJ: Characteriza-
[16] [17] [18]
[19]
[20]
[21] [22]
[23]
[24]
[25]
[26] [27]
[28] [29]
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tion of intercellular adhesion molecule-l and HLA-DR expression in normal and inflamed skin: modulation by recombinant gamma interferon and tumor necrosis factor. J Am Acad Dermatol 20: 617-629, 1989. Schopf RE, Naumann S, Rehder M, Morsches B: Soluble intercellular adhesion molecule-l levels in patients with psoriasis. Br J Dermatol 128: 34-37, 1993. Dustin ML, Springer TA: Role of lympocyte adhesion receptors in transient interactions and cell locomotion. Ann Rev Immunol 9: 27-66, 1991. De Boer OJ, Wakelkamp IM, Pals ST, Claessen N, Bos JD, Das PK: Increased expression of adhesion receptors in both lesional and non lesional psoriatic skin. Arch Dermatol Res 286: 304-311, 1994. Groves RW, Allen MH, Barker JNWN, Haskard DO, MacDonald DM: Endothelial leucocyte adhesion molecule-l (ELAM-1) expression in cutaneous inflammation. Br J Dermatol 124: 117-123, 1991. Dustin ML, Carpen 0, Springer TA: Regulation of locomotion and cell cell contact area by the LFA-1 and ICAM- adhesion receptors. J Immunol 148: 2654-2663, 1992. Kellner I, Konter U, Sterry W: Overexpression of extracellular matrix receptors (VLA-3, 5 and 6) on psoriatic keratinocytes. Br J Dermatol 125: 211-216, 1991. Horrocks C, Duncan JI, Oliver AM, Thomson AW: Adhesion molecule expression in psoriatic skin lesions and the influence of cyclosporin A. Clin Exp Immunol84: 157-162, 1991. Petzelbauer P, Sting1 G, Wolff K, Volt-Platzer B: Cyclosporin A suppresses ICAM- expression by papillary endothelium in healing psoriatic plaques. J Invest Dermato1 96(3): 362-369, 1991. De Pita 0, De Sanctis G, Bottari V, Ruffelli M, Simoni R, Frezzolini A, Benucci R, Fontana L: ICAM- expression on PBL from psoriasis: differences between acute and remission phase. Chron Dermatol 5: 1-9, 1995. King LE, Gates RE, Stoscheck CM, Fava RA, Nanney LB: Role of epidermal growth factor in psoriasis. In: Psoriasis. Edited by HH Roenigk Jr, HI Maibach. Marcel Dekker, New York, 1991, pp. 431-443. Fry L, Thomson AV: Soluble IL-2 receptor and CD25 cells in psoriasis: effects of Cyclosporin A and PUVA therapy. Clin Exp Immunol 85: 293-296, 1991. Shiohara T, Imanishi K, Sagawa Y, Nagashima M: Differential effects of cyclosporine and etretinate on serum cytokine levels in patients with psoriasis. J Am Acad Dermatol 27: 5688574, 1992. Sztaba-Kania M, Placek W: Plasma fibronectin in psoriatic patients. Arch Dermatol Res 287: 219-220, 1995. Fyrand 0: Studies of fibronectin in the skin. II. Indirect immunofluorescence studies in psoriasis vulgaris. Arch Dermatol Res 226, 33-41, 1979.
Dermatological Science Journal of Dermatological Science13 (1996) 118%124
Psoriasis: comparison of immunological markers in patients with acute and remission phase 0. De Piti”, M. Ruffelli”, S. Cadoni”, A. Frezzolini”, G.F. Biavab, R. Simomb, V. Bottari”, G. De Sanctis” “Department ‘Department
of Surgery,
of Dermatology, Division of Immunology, bDepartment of Dermatology,I.D.I.-IRCCS, Clinical Immunology and Allergology ‘Tor
I.D.I.-IRCCS, Rome, Italy Rome, Italy University of Rome, Vergata’,
Rome,
Italy
Received 21 August 1995;revised 7 December 1995;accepted 28 December 1995
Abstract
The immunesysteminvolvement in psoriasishas been documentedby the presenceof activated T-cells both in peripheralblood and in psoriatic skin lesionsand by the intervention of cytokines in the inflammatory process.On this basis,we have undertaken a study in order to examine, in addition to activation markers such as CD25 and CD54 (ICAM-1) on peripheralblood mononuclearcells(PBMNCs) surface,serumlevelsof solubleinterleukin (IL)-2 receptor (sIL-2R), solubleICAM- (sICAM-l), solubleCD4 (sCD4), solubleCD8 (sCD8), p2-microglobulin and fibronectin (FN) in psoriatic patientsanalyzed both in acute and remissionphaseobtainedby topical therapy alone. Our resultsshow that PBMNCs expressingIL-2 receptor (CD25) were increasedboth in percentageand absolute numberin respectto controls, and were not modifiedafter remission.On the contrary, the significantly higher number of CD54+ lymphocytes evaluated in acute psoriasis,showeda reduction during the remissionphase,even if the values persistedhigher than controls. Serum levels of sIL-2R, sICAM-1, sCD4, sCD8 and /YZmicroglobulin were significantly higher than controls both in acute and remissionphase; only FN levels were found to be lower, in patients evaluatedboth in acutepsoriasisand after therapy, in respectto normal donors.On the whole, theseresults seemto indicate the persistenceof both cellular and solubleactivation markerseven in psoriasisremissionphase;in this light, we can supposethat topical therapy alone is not able to efficiently down-regulateactivation mechanisms involved in the pathogenesisof the disease. Keywords:
Psoriasis;Immune system;Solublefactors; Adhesion molecules;Topical therapy
1. Introduction
The relevance of immune and inflammatory factors in the pathogenesis of psoriasis is suggested by the production of cytokines from epi-
dermal keratinocytes even in normal appearing skin, by increased production of inflammatory cytokines from activated T-lymphocytes, and by enhanced functions of dendritic antigen presenting cells [l-8]. In the acute phase, a prevalent
0923-181 l/96/$15.000 1996ElsevierScience IrelandLtd. All rightsreserved PII SO923-1811(96)00517-8
0. De Pith
et al. /Journal
of Dermatological
infiltration of CD4 + and DR + T-lymphocytes is present in the lesional skin, while CD8 + lymphocytes are found in perilesional skin and in remission phase [l]. The presence of soluble immune products such as CD4 and CD8 molecules and of some cytokines in both suction-blister fluid and serum from psoriatic patients accounts for the concomitant involvement of skin-associated and peripheral immune system [9-131. In this context, cytokine-inducible adhesion molecules and some receptor-ligand pairs can regulate lymphocyte traffic in vivo, in relation to both the stage of inflammation and the tissue involvement [14-201. An overexpression of VLA-3, -5, -6 on endothelial cells and the presence of LFA-1 + and ICAM- 1 + infiltrating mononuclear cells have been recently documented in acute psoriasis, while a decrease of the ICAM+ lymphocytes has been shown in the uninvolved skin [21-231. These data strongly support the possibility that circulating lymphocytes could share the same activation markers with skin infiltrating lymphocytes, and our recent work demonstrates a large number of circulating CD54 + lymphocytes in peripheral blood samples from patients with active psoriasis ~241.
In this study we have analyzed, in addition to activation markers on peripheral blood lymphocytes (i.e. CD25 and CD54), serum levels of soluble IL-2 receptor (sIL-2R), soluble ICAM1 (sICAM-l), soluble CD4 (sCD4), soluble CD8 (sCD8), b-microglobulin and fibronectin in patients with acute psoriasis, in order to evaluate the presence of a T-cell activation and homotypic and heterotypic cell-cell adhesion molecule expression. Furthermore, the efficiency of a classical topical treatment on such parameters has been evaluated.
2. Materials and methods 2.1. Patients
Thirty consecutive patients (23 males and 7 females) aging from 20 to 80 years of age, suffering from psoriasis (27 vulgaris and 3 erythrodermic) were studied.
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13 (1996)
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The PAS1 (psoriasis area and severity index) score ranged from 2 to 44 (median 2 1.3). Furthermore patients were analyzed considering the extension of cutaneous involvement (total body surface area, TBSA) excerpted from PAS1 score and thus subdivided in four groups: TBSA < 25% (I stage): 4 patients 25-50% (II stage): 12 patients 50-75% (III stage): 13 patients < 75% (IV stage): 1 patient All patients were evaluated in acute phase and after two weeks of topical therapy alone, using keratolytic agents and/or steroids. We obtained 22 complete remissions (CR) and 8 partial remissions (PR); the mean reduction of cutaneous involvement in PR was 64 + 13%. Blood samples from 11 healthy donors having a similar sex and age distribution, processed at the same time as those of the patients, were used as controls. 2.2. Peripheral blood lymphocytes phenotypical analysis
Peripheral blood specimens were collected in 10 ml vacutainer tubes (Becton-Dickinson, Mountain View, CA) containing ethylenediaminetetraacetate (EDTA). Complete blood counts and leukocytes differential counts were obtained. Preparation of blood cells for flow cytometry was performed by incubating 0.1 ml of blood for 45 min at 0-4°C with monoclonal antibodies (mAbs). mAbs directed against CD3, CD4, CD8, CD19, CD25, CD56 were obtained from Becton-Dickinson, CDlla and CD54 from Immunotech Int., Marseille, France. The samples were added with fats lysing solution, washed twice with phosphate buffer saline (PBS) and then resuspended in PBS. Flow cytometric analysis was performed with a Becton-Dickinson FACScan equipped with an argon ion laser at 15 mW power and filter sets for fluoresceine and phycoeritrin. For each sample, 10 000 events were evaluated. 2.3. Soluble factors
Enzyme-linked immunosorbent assays (ELISA) test kits, utilizing specific murine mAb-coated
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Table 1 PBL phenotypic analysis in controls (n = 11) and in psoriatic patients (n = 30) evaluated in acute phase and in remission Controls
CD19 CD3 CD4 CD8 CD56
Acute phase
%
a.n.
14*5 71 +8 44k6 27 k 7 15*5
283 + 1385 k 858 + 527 k 293 k
116 492 221 158 120
Remission phase
%
a.n.
10*4 70 * 9 42 f 8 28 +9 14*9
226 + 1583 f 950 f 633 f 317 k
93 384 246 185 138
%
a.n.
11+4 71&7 43 + 8 29 k 7 15+9
225 f 98 1453+315 880 f 221 593 + 154 307 f 122
Data are reported as mean values k S.D. both in percentage (%) and in absolute number (a.n.).
polystirene microwells were employed to detect serum levels of sCD4, sCD8, sIL-2R (T-Cell Diagnostics, Inc. Cambridge, USA), sICAM-1 (R and D Systems, Minneapolis, MN, USA) and p2-microglobulin (Farmos Diagnostica, Finland). Fibronectin was detected by a radial immunodiffusion assay (The Binding Site Ltd., Birmingham). All samples or standards were assayed in duplicate. Intra- and inter-assay variability was less than 3%.
number (479 &- 253 mm3 vs. 166 f 102 mm3; P < 0.001) were significantly increased, when compared with normal donors. After therapy, CD54 + circulating lymphocytes decreased in both percentage (15.2 k 6.4%; P < 0.001) and absolute number (320 ) 170 mm3; P < O.OOl), even if they never returned to normal values (Fig. 2).
2.4. Statistical analysis
Serum levels of sIL-2R, during the acute phase, were significantly higher than controls (863 +419 U/ml vs. 584 f 140 U/ml; P <0.02), and were not modified after remission (723 + 506 U/ml) (Fig. 3).
Analysis of significance involved paired and unpaired Student’s two-tailed t-test, and P values less than 0.05 were considered significant.
3.2. Serum levels of siL-2R
3.3. Serum levels of sICAM-1 3. Results 3.1. Phenotypical analysis of PBMCs
In acute psoriasis patients, we found normal levels of CD19+, CD3+, CD4+, CD8+ and CD56+ circulating lymphocytes, which were not modified in the remission phase (Table 1). PBMCs expressing IL-2 receptor (CD25) were increased both in percentage (8.5 + 5.5% vs. 2.7 f 0.8%; P < 0.001) and in absolute number (188 f 145 mm3 vs. 52 + 13 mm3; P
Serum levels of sICAM-1 evaluated in acute phase were significantly higher than normal donors (448 f 170 rig/ml vs. 222 f 63 rig/ml; P < 0.001). After therapy we observed a significant decrease (353 + 92 rig/ml; P < 0.02), even if these values remained above control mean values (P < 0.001) (Fig. 4). 3.4. Serum levels of sCD4 and sCD8
Large amounts of soluble molecules released from activated T-cells, in comparison with normal donors, were also found in sera from patients with acute psoriasis. Levels of both sCD4 (71 + 40 U/ml vs. 29 & 10 U/ml; P < 0.001) and
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Fig. 1. Absolute number and percentage of CD25 + PBL in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
sCD8 (521 + 271 U/ml vs. 279 f 69 U/ml; P < 0.001) were higher than controls and were not reduced after the remission, even if sCD8 showed a trend toward a reduction (418 + 278 U/ml; P = 0.07) (Figs. 5 and 6). 3.5. /32-microglobulin
/32-microglobulin levels, higher than controls (2.9 +_ 1.1 pg/ml vs. 1.3 + 0.3 pg/ml; P < O.OOl), significantly decreased in the remission (2.4 f 0.7; P~0.05) (Fig. 7). 3.6. Fibronectin
Fibronectin was found to be reduced in both acute phase (339 + 112 mg/l vs. 529 + 163 mg/ml; P < 0.001) and remission (320 f 97; P < 0.001) (Fig. 8).
4. Discussion Our results demonstrate that some cellular and soluble activation markers are present in bloodstream of psoriatic patients independent of the disease activity. The rise of cell membrane-associated and soluble form of CD25, as well as the similar feature
observed for sCD4 and sCD8 molecules, suggests a chronic T-cell activation. To this regard, literature data report that T-cell activation may be driven by some proinflammatory cytokines which have been found both in the lesional skin [6,7,9,10,25] and in peripheral blood of psoriatic patients [12] in acute as well as in remission phase. In our view it is likely that such activation markers may result from a prevalent skin immune system involvement and that topical reducent therapy alone is able to improve the cutaneous inflammatory process, but not to modulate the underlying immune mechanism. On the contrary, it has been reported that prolonged systemic treatments (i.e. corticosteroids or cyclosporine) are efficient to down-regulate T-cell activation as demonstrated by the reduction of sIL-2R serum levels [26]. Similarly, the high serum levels of /?2-microglobulin, the invariant chain of MHC-I molecules, reflect the rate of activation and proliferation of cutaneous cells (i.e. keratinocytes, Langerhans, etc.); its persistence at high levels after local therapy further supports the presence of a residual activation of the skin immune system. ICAMoverexpression seems to be related to the acute inflammatory process, as demonstrated by a positive correlation between the number of circulating CD54+ lymphocytes and the extension of the cutaneous involvement [24], and
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Fig. 2. Absolute number and percentage of CD54+ PBL in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001); 0 indicates statistical significance calculated by paired Student’s ‘-test between acute and remission phase (P i 0.05).
the persistence of abnormal numbers of CD54 + circulating lymphocytes in remission phase confirms that cutaneous immunoflogosis is still present. Furthermore, the presence of high levels of sICAM-I even during the remission phase suggests that the rise of adhesion molecules may be related to an inadequate inhibition of up-regulatory inflammatory cytokine production by local therapy alone; also it has been demonstrated that a residual cytokine production is present in psori-
Fig. 3. Serum levels of soluble IL-2 receptor in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Students t-test versus controls (P < 0.02).
atic patients treated with systemic therapy [27]. The activation of adhesive processes could be at least in part responsible for the significantly lower fibronectin serum levels detected in our patients, contrary to recent literature data that report high levels of fibronectin in plasma of psoriatic patients [28]. In our opinion, soluble fibronectin could be recruited into lesional skin by cutaneous cell over-
Fig. 4. Serum levels of soluble ICAM- in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P
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Fig. 5. Serum levels of soluble CD4 in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.) * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
Fig. 7. Serum levels of soluble p2-microglobulin in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001); indicates statistical significance calculated by paired Student’s t-test between acute and remission phase (P < 0.05).
expressing specific receptors, or by circulating lymphocytes and polymorphonuclears which actively carry fibronectin molecules on their cell membrane, subtracting them from peripheral bloodstream. In this regard Fyrand demonstrated the presence of large amounts of fibronectin into lesional skin of patients affected by psoriasis vulgaris [29].
In conclusion, the presence of both cellular and soluble activation markers in the bloodstream during acute psoriasis and remission phases seems to account for a systemic involvement of the disease. However, we can also suppose that topical therapy alone is able to induce only its cosmetic effect, irrespective of an efficient immunomodulation. ) Fibrooectin
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Fig. 6. Serum levels of soluble CD8 in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
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Fig. 8. Serum levels of soluble fibronectin in psoriatic patients. Mean values are indicated by white (controls), lined (patients in acute phase, A.P.) and dotted columns (patients in remission phase, R.P.). * indicates statistical significance calculated by unpaired Student’s t-test versus controls (P < 0.001).
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