STUDY Livedo Vasculopathy vs Small Vessel Cutaneous Vasculitis Cytokine and Platelet P-Selectin Studies Massimo Papi, MD; Biagio Didona, MD; Ornella De Pita`, MD; Alessandra Frezzolini, PhD; Stefano Di Giulio, MD; Walter De Matteis, MD; Domenico Del Principe, MD; Rino Cavalieri, MD Objective: To assess the role of platelets and lymphocyte- both LV (1.24 ± 0.46 IU/mL [mean ± SD] vs 0.46 ± 0.24 related immunological mechanisms in livedo vasculopa- IU/mL, P,.001; 899 ± 368 IU/mL vs 628 ± 132 IU/mL, thy (LV) and cutaneous small vessel vasculitis (CSVV). P<.02) and CSVV (0.91 ± 0.57 IU/mL, P,.02; 1087 ± 451 Livedo vasculopathy is thought to be related to the throm- IU/mL, P,.001) than in those from the healthy con- botic occlusion of small and medium-sized dermal ves- trols. The serum levels of IL-1b, TNF-a, and IL-8 were sels. Cutaneous small vessel vasculitis comprises a het- higher in patients with CSVV than in controls (7.53 ± erogeneous group of disorders in which the main 6.7 pg/mL vs 4.58 ± 2.72 pg/mL; 23.7 ± 12.6 pg/mL vs pathogenetic events could be modulated by circulating 10.82 ± 2.46 pg/mL, P,.001; 37.8 ± 46 pg/mL vs 8.25 ± cytokines. 3.53 pg/mL, P,.02, respectively). No significant differ- ence in the serum levels of IL-1b (7.2 ± 4.9 pg/mL), TNF-a Design: Case series study of 2 groups of patients af- (12.9 ± 3.47 pg/mL), and IL-8 (5.9 ± 4.13 pg/mL) was fected respectively with LV and CSVV. observed in patients with LV compared with controls. An increased expression of platelet P-selectin was also de- Setting: A large clinical and research institute for the tected in patients with LV in comparison with controls study and treatment of cutaneous diseases. and patients with CSVV. The mean ± SD percentage of positive cells for P-selectin was 43% ± 5% in the pa- Patients: Consecutive patients with clinically and tients with LV, 5.1% ± 2% in the controls (P,.001), and histologically proved idiopathic LV (n = 8) and CSVV 5.3% ± 2% in the patients with CSVV (P,.001). (n = 20) were studied and compared with healthy donors (n = 20). Patients with potentially correlated Conclusions: Taken together, these data demonstrate systemic diseases were excluded. that different pathogenetic mechanisms are operative in LV and CSVV. In fact, platelet and lymphocyte activa- Main Outcome Measures: Surface expression of plate- tion is present in LV, whereas the levels of inflamma- let P-selectin and circulating level of interleukin (IL) 1b, tory mediators are in a normal range. In CSVV, the high tumor necrosis factor a (TNF-a), IL-8, IL-2, and soluble serum levels of proinflammatory cytokines suggest that IL-2 receptor. they are actively involved in the pathogenesis of cutane- ous vasculitis. Results: The IL-2 and soluble IL-2 receptor levels were significantly higher in serum samples from patients with Arch Dermatol. 1998;134:447-452 IVEDO vasculopathy (LV) is cemosa (Figure 1). Histologically, it an uncommon clinicopatho- shows segmental hyalinization, endothe- logic condition also known lial swelling, and thromboses of dermal 1,2 From the Departments of as livedo vasculitis, atro- vessels (Figure 2). A variable number of Dermatology (Drs Papi, phie blanche,3 and PURPLE perivascular lymphocytes are usually ob- Didona, and Cavalieri) and (painful purpuric ulcers with reticular pat- L 4 Immunodermatology tern of the lower extremities) in which the For editorial comment (Drs De Pita` and Frezzolini), histological finding of leukocytoclasia is Istituto Dermopatico usually absent. It is clinically character- see page 491 Immacolata, Istituto Ricovero e ized by early, focal, painful purpuric le- Cura a Carattere Scientifico, and the Department of sions of the lower extremities that fre- served. The prevalence is estimated around Pediatrics, Universita` Tor quently ulcerate and slowly heal, leaving 1:100 000 per year and is more frequent 5 Vergata (Drs Di Giulio, white, stellate, atrophic scars (atrophie in young women (1:3). On the basis of De Matteis, and Del Principe), blanche), telangiectasias, hemosiderinic li- the clinical evolution, the improvement Rome, Italy. vedolike hyperpigmentation, and livedo ra- during fibrinolytic and anti- ARCH DERMATOL / VOL 134, APR 1998 447 ©1998 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 PATIENTS AND METHODS protein at 1200g for 15 minutes produced a platelet- containing pellet, which was washed and resuspended in phosphate-buffered saline (pH, 7.4). PATIENTS IMMUNOENZYMATIC ASSAY The study included 8 patients (5 women and 3 men; mean age, 32.5 years) showing the typical clinical and histologi- Serum levels of IL-1b, TNF-a, IL-8, IL-2, and sIL-2R were cal features of LV and 20 patients (12 women and 8 men; measured by enzyme-linked immunosorbent assay test kits mean age, 46 years) with clinical and histological mani- (Medgenics Diagnostic, Fleurus, Belgium [IL-1b, TNF-a, festations of CSVV who were hospitalized at Istituto Der- and IL-2]; R&D Systems, Minneapolis, Minn [IL-8]; and mopatico Immacolata, Rome, Italy, during the last 4 years. Cell Free, T cell Science, Cambridge, Mass [sIL-2R]). All Twenty healthy donors matching the patients in age and assays were based on the dual immunometric sandwich prin- sex distribution were evaluated as a control group. We ex- ciple and were performed according to the manufacturer’s cluded all patients with both LV and CSVV and poten- instructions. tially related systemic diseases. The LV lesions were ex- clusively localized on the lower part of the legs and the FLOW CYTOMETRIC ANALYSIS dorsum of the feet, whereas the CSVV purpuric manifes- tations involved not only the legs but also, in some cases, A 1-mL platelet suspension containing 5310 cells was in- the thighs and glutei. No patients were receiving therapy cubated in the dark with 20 µL of anti–P-selectin fluores- when a blood sample and a skin biopsy specimen of a re- cein isothiocyanate–labeled monoclonal antibody (mAb) cent active lesion were obtained. Laboratory and immu- for 30 minutes at room temperature. At the end of incu- nological studies, including determinations of antinuclear bation, the platelets were washed 3 times in phosphate- antibodies, antibodies to DNA, antiendothelial antibod- buffered saline. Nonspecific binding of mAb was assessed ies, antineutrophil cytoplasmic autoantibodies, anticardio- by incubating platelets with fluorescein isothiocyanate– lipin antibodies, lupus anticoagulant, serum complement labeled anti-CD13 mAb. The results of fluorescence were levels (C3, C4, and CH5M0), circulating immune com- analyzed in a flow cytometer (FACscan, Becton-Dickin- plexes, cryoglobulins, cryofibrinogen, rheumatoid factor, son, Rutherford, NJ). All measurements were monitored and circulating lymphocyte subpopulations, were rou- after excitation of cells passing through the beam of an air- tinely performed in all patients. cooled argon ion laser (488-nm excitation,15-mW out- All patients consented to participate in the study, which put). Parameters collected included forward-angle light scat- was approved by the Ethical Committee of the institution. ter (FSC), 90° light scatter, and 530-nm green fluorescence. Representative clinical and histological findings of LV are A gate was set around the platelets, and 5000 cells were shown in Figures 1 and 2 and the typical clinical appear- analyzed for fluorescein isothiocyanate fluorescence to quan- ance and histological signs of CSVV are shown in Figures tify the amount of the platelet-bound mAb. The binding 3 and 4. of the antibody was expressed as the percentage of plate- lets positive for antibody. Anti–P-selectin (platelet activa- SERUM SAMPLES tion–dependent granule external membrane [PADGEM]) (IgG1 and mouse) and anti–CD13 (panmyeloid, IgG1, and Serum samples were collected and stored frozen at −80°C mouse) fluorescein isothiocyanate–labeled mABs were com- until assayed for cytokines. For flow cytometric studies, the mercially obtained (Immunotech, Marseille, France). platelets were prepared from blood samples obtained from an antecubital vein through a 19-gauge butterfly needle with STATISTICAL ANALYSIS no tourniquet in an attempt to minimize platelet activa- tion during blood collection. After the first 2 mL of blood Statistical analysis was performed using a Student t test for was discharged, 4.5 mL was collected into a plastic sy- unpaired data, and P values of less than .05 were consid- ringe containing 200 µL of 1% glutaraldehyde solution and ered significant. The analysis of the fluorescence intensity edetic acid (final concentration, 5 mmol/L). Platelet-rich histogram was performed using the Kolmogorov-Smirnov plasma was obtained by centrifugation of blood at 100g for 2-sample test. Only values of a,.001 were considered sig- 15 minutes. Further centrifugation of platelet-rich nificant. platelet therapy,6-8 and the detection of elevated serum cryoglobulinemia, and septic vasculitis). They refer to spe- fibrinopeptide A levels,2 LV is thought to be mediated cific inflammatory changes of venules, arterioles, and cap- by a vasocclusive mechanism. Nevertheless, specific his- illaries. A pathogenetic role of circulating immune com- tological findings, such as thickened blood vessel walls, plexes and autoantibodies (eg, antineutrophil and occasional deposition of immunoreactants, and fibrin,9 antiendothelial antibodies) in these syndromes is dif- and the possible association with immune-mediated dis- fusely accepted. The skin is a preferential target organ eases suggest a potential pathogenetic role of immuno- of SVV mostly resulting in clinical features of palpable logical mechanisms. Tissue plasminogen activator treat- purpura (Figure 3) and histological changes of leuko- ment was recently reported to be effective in LV.10 cytoclastic vasculitis (Figure 4). Cutaneous SVV (CSVV) Small vessel vasculitis (SVV), according to Joriz- can be limited to the skin or can represent the cutane- zo’s11 recent classification of vasculitis, includes a wide ous involvement in the case of systemic vasculitis.