Hindawi Mediators of Inflammation Volume 2019, Article ID 2473164, 12 pages https://doi.org/10.1155/2019/2473164 Research Article Association of Platelet Binding to Lymphocytes with B Cell Abnormalities and Clinical Manifestations in Systemic Lupus Erythematosus Carlos Zamora ,1 Elide Toniolo,2 Cesar Diaz-Torné,2 Elisabet Cantó,1 Berta Magallares,2 Ma Angels Ortiz,1 Lidia Perea,1 Hector Corominas,2 and Silvia Vidal 1 1 Department of Immunology, Biomedical Research Institute Sant Pau (IBB Sant Pau), Barcelona, Spain 2 Unit of Rheumatology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Correspondence should be addressed to Carlos Zamora; czamora@santpau.cat and Silvia Vidal; svidal@santpau.cat Received 25 September 2018; Revised 28 December 2018; Accepted 6 January 2019; Published 3 March 2019 Academic Editor: Emiliano Antiga Copyright © 2019 Carlos Zamora et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Systemic lupus erythematosus (SLE) is an autoimmune disease associated with the polyclonal activation of B lymphocytes and the production of autoantibodies that cause immune complex-related inflammation. Immunological factors derived from platelets modulate B cell function in SLE disease. However, platelets do not only modify the immune system by soluble factors. The binding of platelets to lymphocytes can modulate immune response. Thus, we speculate that the binding of platelets to lymphocytes in SLE patients may play a role in abnormal B lymphocyte response and the pathogenesis of SLE. We observed that levels of lymphocytes with bound platelets were higher in SLE patients than in healthy donors (HD). In SLE patients, the percentage of B lymphocytes with bound platelets positively correlated with plasmatic levels of IgG, IgA, IL-10, and soluble CD40L and negatively correlated with IgM levels, though not in HD. Preswitched memory B lymphocytes were the subpopulation with more bound platelets. Lymphocytes with bound platelets from both HD and SLE patients had major levels of CD86 and BAFFR and a greater production of IL-10 than lymphocytes without bound platelets. However, only B lymphocytes with bound platelets from SLE patients had increased levels of IgG and IgA on their surface. SLE patients with a suggestive renal manifestation had the highest levels of B and T lymphocytes with bound platelets. These results suggest that the binding of platelets to lymphocytes plays a role in SLE disease and that controlling this binding may be a promising therapeutic approach. 1. Introduction disease activity (SLEDAI) and the production of anti- dsDNA antibodies [10]. IL-10 has a positive influence on B SLE has been associated with polyclonal B cell hyperactivity cell survival, proliferation, differentiation, and autoantibody and autoantibody production [1–3]. B lymphocytes from production [9]. Blocking IL-10 in SLE patients decreases SLE patients also display an increased expression of the autoantibody production and reduces clinical symptoms activated markers CD86, CD80, and CD38 [4, 5] and IgG and disease activity [11, 12]. This IL-10 overproduction in and IgA [6] levels on their surface. These patients have an SLE patients has been ascribed to B lymphocytes and mono- elevated proportion of memory B lymphocytes and a cytes [13]. Since B regulatory lymphocytes are the major B decreased proportion of preswitched B lymphocytes [7, 8]. lymphocytes producing IL-10, they likely contribute to SLE IL-10 is an anti-inflammatory cytokine secreted by Tregs, pathogenesis [14, 15]. Bregs, Th2 lymphocytes, monocytes, and other cells that Platelet has recently been recognized as an immunoregu- plays a crucial role in preventing inflammatory and autoim- latory cellular component [16]. Upon activation, platelets mune disease [9]. However, there is an elevated concentra- release cytokines, chemokines, growth factors, and platelet- tion of plasma IL-10 in SLE and these levels correlate with derived microparticles (PMP) and express a set of activation 2 Mediators of Inflammation molecules on their membrane (P-selectin and CD40L) that [36]. Two of the 21 patients did not meet to the Systemic allow the platelet binding to leukocytes [17–20]. Platelets Lupus International Collaborating Clinics (SLICC) criteria can bind to lymphocytes and P-selectin-PSGL-1 appears to for SLE classification. Patients previously treated with B be essential in this interaction. However, other molecules, cell-targeted therapies, with other biologic agents, or with such as CD40-CD40L, GPIb-CD11b, and GPIIb/IIIa-CD11/ more than 10 mg per day of prednisone were excluded. To CD18, can also play a role in the binding of platelet to lym- assess disease activity, patients’ Systemic Lupus Erythema- phocytes [21]. Platelets directly modulate humoral activity, tosus Disease Activity Index (SLEDAI) was calculated at stimulating B cell proliferation, antibody production, and the time of the sample collection [37]. In Table 1, we the membrane expression of CD27 and CD86, via soluble show the demographic, clinical, and laboratory data of CD40L (sCD40L) [22, 23]. Both mechanisms, platelet- SLE patients enrolled in this study. Written informed con- derived soluble factors (TGFβ, PF4, and sCD40L) and the sent was obtained, and ethical approval for the study was binding of platelets to leukocytes (CD62P-PSGL1), decrease granted by the Hospital de la Santa Creu i Sant Pau Institu- T cell proliferation and inflammatory cytokine production tional Ethics Committee. and increase IL-10 production by T lymphocytes and mono- Peripheral blood mononuclear cells (PBMCs) were iso- cytes [18–20, 24, 25]. Through CD40-CD40L ligation, plate- lated by Ficoll-Hypaque density gradient (Lymphoprep, lets support B cell isotype switching [23, 26] and, with the AXIS-SHIELD PoCAs, Oslo, Norway). collaboration of Th cells, platelets enhance germinal center formation [27]. In fact, CD40L-deficient mice failed to elicit 2.2. Staining of Peripheral Blood Mononuclear Cells and B cell isotype switch and this failure could be corrected by Whole Blood Cells. PBMCs (1 × 106 cells) were incubated infusing CD40L-expressing platelets or platelet-free superna- with anti-CD19-PEDy647, anti-CD38-PE, anti-CD41a-FITC, tant from activated platelets containing sCD40L [26]. Simi- anti-CD41a-PE (Immunotools, Friesoythe, Germany), anti- larly, the blocking of CD40L in cocultures of platelets with CD19-PECy7, anti-CD27-APC, anti-BAFFR-FITC, anti- B lymphocytes partially abolishes the binding of platelets to TACI-PE (BioLegend, San Diego, USA), anti-CD86-PE, B lymphocytes [23]. In SLE patients, high levels of plasma anti-IgG-PE, anti-IgD-FITC (BD), anti-IgA-FITC, and anti- sCD40L are observed, which are associated with disease IgM-PE (Dako, CA, USA) mAbs and the corresponding severity and the development of nephritic lupus [28, 29]. isotype controls. Whole blood (100 μl) was incubated with sCD40L is released from platelet surface after its activation, anti-CD4-PECy7 (BioLegend), CD5-PE (BD Biosciences), and it is considered an in vivo marker of platelet activation CD19-PEDy647, and CD41a-FITC (Immunotools). Red [30, 31]. However, since CD40L is overexpressed on SLE T blood cells were lysed, and white cells fixed using BD FACS lymphocytes [28, 32, 33], and it can be shed from the mem- lysing solution (BD Biosciences) to be analyzed by flow brane [34]; plasmatic sCD40L in SLE patients is not the sole cytometry. product of platelets. An elevated number of circulating lymphocyte-platelet complexes and an increased platelet 2.3. Flow Cytometry Analysis of Surface Markers and IL-10 activation have been reported in patients with SLE and other Production. We were able to identify the main T and B lym- autoimmune diseases [19, 35]. However, the binding of phocyte subsets combining anti-CD19, anti-CD4, anti-CD8, platelets to different lymphocyte subpopulations in SLE and anti-CD5. Two subsets of B lymphocytes with bound patients has not yet been studied and there is a lack of infor- platelets (PLTs) were identified as CD19+CD5-CD41a+ mation about the relationship between platelet-binding to (CD19+PLT+) and CD19+CD5+CD41a+, respectively. CD4+ lymphocytes, lymphocyte function, and the clinical outcome and CD8+ T lymphocytes with bound platelets were of these patients. identified as CD19-CD4+CD5+CD41a+ (CD4+PLT+) and Our aim was to analyze the possible role of lymphocyte- CD19-CD4-CD5+CD41a+ (CD8+PLT+), respectively, on platelet complexes in the altered B cell function and SLE gated lymphocytes. Activation markers and IL-10 pro- pathogenesis. Firstly, we compared the numbers of lympho- duction were analyzed on CD19+PLT+ and CD19+PLT- cytes with bound platelets in SLE patients and healthy (B lymphocytes without bound platelets). CD19+ and donors. Secondly, we established their relationship with CD19- lymphocytes that secreted spontaneously IL-10 were platelet activation and the isotype class of autoantibodies determined using IL-10 secretion assay (Miltenyi Biotec, and we investigated the phenotype and function of B Bergisch Gladbach, Germany), a kit that measures IL-10 lymphocytes with bound platelets in SLE patients. Finally, secretion on the cell surface. Briefly, after 4 h of resting, we established the relationship between lymphocytes with PBMCs were labeled with IL-10-specific catch reagent on bound platelets and the clinical (hematuria and SLEDAI) and their surface and incubated for 45 min at 37°C under slow laboratory (anti-dsDNA, complement C3, and albumin/ rotation. Cells were washed and labeled with IL-10-PE creatinine) features of patients. detection antibody, CD19-PECy7 (BioLegend), CD41a- FITC (Immunotools), and viability marker LIVE/DEAD 2. Material and Methods fixable Violet Dead Cell Stain kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Then cells were washed, 2.1. Study Subjects and Sample Collection. Whole blood from and membrane IL-10 was analyzed by flow cytometry. Per- 16 healthy donors (HD) and 21 SLE patients was collected in centages of viable lymphocytes did not differ between HD BD vacutainer tubes containing heparin (BD, Franklin Lakes, and SLE patients (90-95%). The percentage of CD19+ B NJ). SLE diagnosis was based on 1982 revised ACR criteria lymphocyte subpopulations with or without bound platelets Mediators of Inflammation 3 Table 1: Demographic, clinical, and laboratory characteristic data of study patients. SLE patients HD General conditions Age; year, mean ± SD 50 67 ± 12 58 49 06 ± 9 26 Gender (%) (n) women 100 (21) 75 (12) Years of evolution, mean ± SD 12 41 ± 10 29 SLEDAI; mean (range) 2.62 (0-12) Laboratory parameters ESRa (mm/h, mean ± SD) 23 85 ± 20 02 CRP (mg/l, mean ± SD) b 2 63 ± 3 48 Complement C3 (mg/dl, mean ± SD) 87 93 ± 20 86 Complement C4 (mg/dl, mean ± SD) 14 12 ± 4 49 Decreased C3 or C4 (%) (n) 80.95 (17) Anti-dsDNA (U/ml), mean (range) 74.8 (22-300) Positive anti-dsDNA (%) (n) 42.8 (9) Positive ANA (%) (n) 95.23 (20) Positive anti-Sm (%) (n) 4.7 (1) Positive antiphospholipid antibody (%) (n) 14.28 (3) Leukopenia and/or lymphopenia (%) (n) 14.28 (3) Clinical features Renal involvement (%) (n) 23.8 (5) Hematuria 23.8 (5) Albumin/creatinine > 2 5 23.8 (5) Cutaneous involvement (%) (n) 28.57 (6) Oral or nasal ulcers 4.7 (1) Alopecia 4.7 (1) Photosensitivity 23.8 (5) Butterfly erythema 9.5 (2) Arthritis (%) (n) 19.1 (4) Nervous system disorder 4.7 (1) Treatment (%) (n) None 9 (2) Mycophenolate 14 (3) Prednisone 48 (10) Hydroxychloroquine 62 (13) Azathioprine 24 (5) Tacrolimus 9 (2) NSAIDc 14.28 (3) Aspirin 23.8 (5) a ESR: erythrocyte sedimentation rate. bCRP: C-reactive protein. cNSAID: nonstereoidal anti-inflammatory drugs. were defined as follows: naïve B lymphocytes (CD27-IgD+ we determined the percentages of cells and the geometric CD41+ and CD27-IgD+CD41-, respectively), preswitched mean fluorescence intensity (MFI) of BAFFR. memory B lymphocytes (CD27+IgD+CD41a+ and CD27+ IgD+CD41a-, respectively), postswitched memory B lym- 2.4. Quantification of Anti-dsDNA, Complement C3, phocytes (CD27+IgD-CD41a+ and CD27+IgD-CD41a-, Albumin/Creatinine, Hematuria, IgG, IgM, and IgA. Anti- respectively), and double-negative (DN) memory B lym- dsDNA levels were determined in serum using Unicap Elia phocytes (CD27-IgD-CD41a+ and CD27-IgD-CD41a-, (Phadia Laboratory Systems, Uppsala, Sweden). Levels of respectively) [38]. Samples were acquired with the MACS- complement C3 in serum and plasma IgG, IgM, and IgA Quant Analyzer 10 flow cytometer (Miltenyi Biotec), and were determined using the Nephelometry System (Beckman 4 Mediators of Inflammation CD19+ CD4+ CD8+ 102 102 102 101 101 101 10 0 10 0 100 HD 4.62 3.8 3.44 10−1 10−1 10−1 15 ⁎⁎ ⁎⁎⁎ ⁎⁎ −2 −2 10 10 10−2 CD19 CD4 CD8 −1 0 1 2 −1 0 1 2 −1 0 1 2 10 10 10 10 10 10 10 10 10 10 10 10 102 102 102 10 % PLT+ 101 101 101 100 100 100 SLE 14.9 11.8 13.7 5 10−1 10−1 10−1 10−2 10−2 10−2 10−1 100 101 102 10−1 100 101 102 10−1 100 101 102 0 CD41a CD41a CD41a CD19 + SLE CD4 + HD CD4 + SLE CD8 + HD CD8 + SLE CD19 + HD 102 102 102 101 101 101 CD19 CD4 CD8 10 0 10 0 100 0 0 0.02 10−1 10 −1 10−1 10−2 10−2 10−2 10−1 100 101 102 10−1 100 101 102 10−1 100 101 102 Isotype CD41a Isotype CD41a Isotype CD41a (a) (b) Figure 1: Percentage of B and T lymphocytes with bound platelets in HD and SLE patients. (a) A representative image of B lymphocytes (CD19+), CD4+ T lymphocytes (CD19-, CD4+, and CD5+) and CD8+ T lymphocytes (CD19-, CD4-, and CD5+) with bound platelets (PLTs) in HD and SLE patients is shown. (b) The dot plot shows the percentage of B and CD4 and CD8 T lymphocytes with bound platelets in HD (n = 16) and SLE patients (n = 21). CD4 and CD8 T lymphocytes had more bound platelets than B lymphocytes in SLE patients (both p < 0 01), but not in HD. Data are presented as the mean. Statistical analysis was performed using the t-test for comparison between HD and SLE and paired t-test for the subpopulation comparisons in HD and SLE. ∗∗ p < 0 01 and ∗∗∗ p < 0 0001. Coulter). Albumin/creatinine was determined in serum by 3. Results the turbidimetry system (Abbott, Chicago, USA). The presence of hematuria was determined by the Combur 3.1. Circulating Lymphocytes with Bound Platelets in SLE test (Roche, Basilea, Switzerland). Anti-dsDNA antibodies Patients. Higher percentages of circulating B and T lympho- were considered positive when levels were >17 UI/ml. cytes with bound platelets were observed in SLE patients than Levels of albumin/creatinine were expressed as mg/mmol. in HD (Figures 1(a) and 1(b)). The percentages of bound Levels of complement C3 and immunoglobulins were platelets to the CD19+CD5+ B cell subpopulation in HD expressed as mg/dl. and SLE patients were comparable (7 15 ± 1 04 and 9 98 ± 1 54, respectively). In SLE patients but not in HD, T lympho- 2.5. Determination of IL-10 and sCD40L. Plasma concentra- cytes had more bound platelets than B lymphocytes (p < 0 01 tions of IL-10 (Immunotools) and sCD40L (Peprotech, for both CD4+ and CD8+ T lymphocytes). The percentages London, UK) were determined using specific ELISA kits of B, CD4+, and CD8+ T lymphocytes with bound platelets according to the manufacturers’ instructions and using the correlated strongly to each other (r = 0 92, p < 0 0001 for specific standard curves of recombinant molecules. The %CD19+PLT+ vs. CD4+PLT+; r = 0 87, p < 0 0001 for limits of detection were as follows: 16 pg/ml for IL-10 and %CD19+PLT+ vs. CD8+PLT+; and r = 0 91, p < 0 0001 31.25 pg/ml for sCD40L. for %CD4+PLT+ vs. CD8+PLT+). The percentage of B and T lymphocytes with bound platelets was not affected 2.6. Statistics. The Kolmogorov-Smirnov test was applied to by the absolute numbers of lymphocytes and platelets (data test the data for normal distribution. Normally distributed not shown). variables were then reported as mean ± s e m. Comparisons between groups were tested with Student’s t-test, the 3.2. Association of B Lymphocytes with Bound Platelets with Mann-Whitney test, or the Wilcoxon test according to the Levels of Immunoglobulin and Platelet Activation. The Gaussian distribution. ANOVA was used to compare percentage of CD19+PLT+ correlated positively with con- more than two groups with normal distributions. Correla- centrations of IgG and IgA levels and correlated negatively tion analyses were carried out with Pearson’s correlation. with concentrations of IgM in SLE patients (Figures 2(a)– p values < 0.05 were considered significant. 2(c)), though not with HD (r = 0 22, p = 0 52 for IgM; Mediators of Inflammation 5 15 15 15 % CD19+ PLT+ % CD19+ PLT+ % CD19+ PLT+ 10 10 10 5 5 5 0 0 0 0 100 200 300 400 500 0 1000 2000 3000 4000 0 200 400 600 800 IgM (mg/dl) IgG (mg/dl) IgA (mg/dl) r = 0.48 r = 0.44 r = 0.59 p = 0.02 p = 0.05 p = 0.006 (a) (b) (c) 15 15 15 % CD19+ PLT+ % CD4+ PLT+ % CD8+ PLT+ 10 10 10 5 5 5 0 0 0 1 10 100 1000 10000 1 10 100 1000 10000 1 10 100 1000 10000 sCD40L (pg/ml) sCD40L (pg/ml) sCD40L (pg/ml) r = 0.61 r = 0.62 r = 0.55 p = 0.006 p = 0.004 p = 0.01 (d) (e) (f) Figure 2: Association of lymphocytes with bound platelets with immunoglobulin’s isotypes and platelet activation in SLE patients. Correlation of CD19+PLT+ percentages with levels (mg/dl) of (a) IgM (b) IgG and (c) IgA in 20 SLE patients. Measurement of Igs in one SLE patient could not be performed due to the lack of sample. Correlation of sCD40L levels (pg/ml) with percentage of (d) CD19+PLT+, (e) CD4+PLT+, and (f) CD8+PLT+ in 19 SLE patients. Measurements of sCD40L in two SLE patients could not be performed by the lack of samples. Pearson’s correlation was performed for the correlation analysis. r = 0 08, p = 0 81 for IgA; and r = 0 31, p = 0 39 for IgG). A the different B lymphocyte subpopulations had a similar positive correlation between the percentage of CD19+PLT+ percentage of cells with bound platelets. However, in SLE and the IgG/IgM ratio was found in SLE patients (r = 0 56, patients, preswitched memory B lymphocytes showed a p = 0 01) but not in HD. No differences in the plasma con- higher percentage of CD19+PLT+ lymphocytes compared centrations (mg/dl) of IgG, IgA, and IgM were observed with the other B lymphocytes subpopulations (p < 0 0001 between HD and SLE patients (IgG: 1083 ± 34 74 for HD by ANOVA) (Figures 3(a) and 3(b)). vs. 1348 ± 125 for SLE; IgA: 262 2 ± 42 85 for HD vs. 319 ± No differences in the percentages of B lymphocytes with 34 39 for SLE; IgM: 129 9 ± 16 55 for HD vs. 149 6 ± 20 76 bound platelets were found between the whole blood and for SLE). Plasma levels of sCD40L in SLE patients were PBMC analysis of HD (5 31 ± 0 54 vs. 6 06 ± 0 85, respec- higher than in HD (50 58 ± 25 79 pg/ml for HD vs. 691 3 ± tively) and SLE (7 6 ± 1 04 vs. 8 15 ± 1 29, respectively). 268 7 pg/ml for SLE, p < 0 01) and correlated with the percentage of each subpopulation of lymphocytes with 3.4. Phenotype of B Lymphocytes with Bound Platelets in HD bound platelets in SLE patients (Figures 2(d)–2(f)), though and SLE Patients. Only in SLE patients, CD19+PLT+ lym- not in HD (r = 0 18, p = 0 66 for CD19+PLT+; r = 0 5, p = phocytes had higher percentages of IgG+ and IgA+ cells 0 25 for CD4+PLT+; and r = 0 28, p = 0 49 for CD8+PLT+). than CD19+PLT- lymphocytes (Figures 3(c) and 3(d)). sCD40L also correlated with IgA levels in SLE patients In HD and SLE patients, CD19+PLT+ lymphocytes had (r = 0 52, p = 0 02). higher percentages of CD86+ cells and expressed higher levels of BAFFR than the counterpart CD19+PLT- lympho- 3.3. Different Binding of Platelet to Preswitched Memory B cytes (Figures 3(e) and 3(f)). However, IgM, CD38, and Lymphocytes in SLE Patients. As expected [7, 8], SLE patients TACI expressions were comparable on CD19+PLT+ and had a higher percentage of DN memory B lymphocytes but a CD19+PLT- lymphocytes from SLE patients and HD (data lower percentage of preswitched memory B lymphocytes not shown). than HD (DN memory: 22 62 ± 2 52% for SLE vs. 12 76 ± 2 26% for HD, p < 0 05; preswitched memory: 3 31 ± 0 73% 3.5. IL-10 Production by B Lymphocytes with and without for SLE vs. 10 85 ± 1 91% for HD, p < 0 01). The percentages Bound Platelets. After 4 h of resting in medium, CD19+PLT+ of naïve and postswitched memory B lymphocytes were lymphocytes contained higher percentages of IL-10+ cells comparable in SLE patients and HD. In the latter group, than CD19+PLT- lymphocytes in HD and SLE patients 6 Mediators of Inflammation Pre-switched 10 2 Post-switched 25.5 Pre-switched 12.2 10 2 ⁎⁎⁎ 30 40 1 ⁎ 10 1 10 HD 30 % CD19+ PLT+ 0 10 10 0 20 5.8 % IgG −1 DN memory Naive 10 10 −1 20 8.96 53.4 −2 10 10 CD19 −1 CD27 0 1 2 −1 0 1 2 10 10 10 10 10 10 10 10 10 2 Post-switched Pre-switched 2 10 34.6 2.33 10 1 0 0 1 10 HD SLE HD SLE HD SLE HD SLE 10 PLT+ PLT− PLT+ PLT− 0 SLE 0 10 10 22.0 CD19+ HD CD19+ SLE 10 −1 Naïve Pre- Post- DN −1 10 DN memory Naive switched switched memory 19.3 43.7 −2 10 −1 0 1 2 −1 0 1 2 10 10 10 10 10 10 10 10 IgD CD41a (a) (b) (c) ⁎⁎ ⁎ ⁎⁎ ⁎ ⁎⁎ 50 25 5 40 20 4 MFI BAFFR % CD86 30 15 3 % IgA 20 10 2 10 5 1 0 0 0 PLT+ PLT− PLT+ PLT− PLT+ PLT− PLT+ PLT− PLT+ PLT− PLT+ PLT− CD19+ HD CD19+ SLE CD19+ HD CD19+ SLE CD19+ HD CD19+ SLE (d) (e) (f) Figure 3: Phenotype and subpopulations of B lymphocytes with bound platelets. (a) A representative image of naïve, preswitched, postswitched, and a double-negative (DN) memory and preswitched memory B lymphocytes with bound platelets from HD and SLE patients. (b) Percentages of CD19+PLT+ in each B lymphocyte subpopulation in HD (n = 6) and SLE (n = 8). Multiple comparisons were analyzed by one-way ANOVA (p < 0 0001 in SLE patients). Percentage of (c) IgG, (d) IgA, (e) CD86, and (f) BAFFR MFI on CD19+PLT+/PLT- from HD (n = 6) and SLE (n = 10). Data are presented the as mean. Statistical analysis was performed using the Mann-Whitney test for unpaired samples and Wilcoxon test for paired samples. ∗ p < 0 05, ∗∗ p < 0 01, and ∗∗∗ p < 0 001. (Figure 4(a)). In SLE patients, we observed a tendency to a CD4+PLT+ and CD8+PLT+ than those with normal levels higher percentage of CD19+PLT+IL10+ lymphocytes than (85-193 mg/dl) (Figure 5(c)). SLE patients with albumin/ in HD (Figure 4(b)). No differences in the percentage of creatinine levels > 2 5 mg/mmol showed higher percentages IL-10+ cells were observed in CD19- lymphocytes in HD and of CD19+PLT+, CD4+PLT+, and CD8+PLT+ than those SLE patients (data not shown). with normal levels (≤2.5 mg/mmol) (Figure 5(d)). SLE We then analyzed the association of plasma IL-10 patients with SLEDAI > 3 (patients with active disease sug- concentration with platelet activation, measured as sCD40L gestive of treatment change) [39, 40] showed higher percent- concentration, and with the percentage of circulating lym- ages of CD19+PLT+, CD4+PLT+, and CD8+PLT+ than phocytes with bound platelets. In SLE patients, IL-10 con- those with a SLEDAI ≤ 3 (CD19+PLT+: 8 51 ± 0 57 for >3 centration correlated with sCD40L levels and with the vs. 6 43 ± 0 49 for ≤3, p = 0 02; CD4+PLT+: 9 73 ± 0 66 for percentages of CD19+PLT+, CD4+PLT+, and CD8+PLT+ >3 vs. 7 11 ± 0 64 for ≤3, p = 0 02; and CD8+PLT+: 9 49 ± cells (Figures 4(c)–4(f)), but not in HD (r = 0 33, p = 0 59 for >3 vs. 7 22 ± 0 6 for ≤3, p = 0 03). Comparable per- 0 41 for sCD40L; r = 0 58, p = 0 12 for CD19+PLT+; r = centages of each subpopulation of lymphocytes with bound 0 52, p = 0 18 for CD4+PLT+, and r = 0 67, p = 0 067 for platelets were observed when SLE patients were segregated CD8+PLT+). according to medication and cutaneous or articular manifes- tations (data not shown). 3.6. Relationship between Lymphocytes with Bound Platelets and Clinical Features. SLE patients with positive anti- 4. Discussion dsDNA titers and those with hematuria had higher percent- ages of each subpopulation of lymphocytes with bound We found that the percentages of B lymphocytes with platelets than SLE patients with negative anti-dsDNA titers bound platelets were increased in SLE patients and corre- and without hematuria (Figures 5(a) and 5(b)). No correla- lated with plasma levels of sCD40L, IL-10, IgG, IgA, and tion was found between the levels of anti-dsDNA antibodies IgM. B lymphocytes with bound platelets had upregulated and the percentage of CD19+PLT+. SLE patients with low activation markers and IL-10 production when compared complement C3 levels (<85 mg/dl) showed a higher percent- to B lymphocytes without bound platelets. The highest age of CD19+PLT+ and a tendency to higher percentages of percentages of lymphocytes with bound platelets were Mediators of Inflammation 7 ⁎ ⁎⁎ % CD19+ IL10+/ %CD19+ 5 1 10000 0.09 4 % CD19+ IL10+ pg/ml sCD40L 1000 0.1 3 100 2 0.01 1 10 0 0.001 1 PLT+ PLT− PLT+ PLT− HD SLE HD SLE 1 10 100 1000 10000 CD19+ HD CD19+ SLE CD19+ PLT+ CD19+ PLT− IL-10 (pg/ml) r = 0.75 p < 0.001 (a) (b) (c) 15 15 15 % CD19+ PLT+ % CD4+ PLT+ % CD8+ PLT+ 10 10 10 5 5 5 0 0 0 1 10 100 1000 10000 1 10 100 1000 10000 1 10 100 1000 10000 IL-10 (pg/ml) IL-10 (pg/ml) IL-10 (pg/ml) r = 0.41 r = 0.48 r = 0.46 p = 0.03 p = 0.01 p = 0.01 (d) (e) (f) Figure 4: Association of B lymphocytes with bound platelets and IL-10 production and plasma levels. (a) Percentage of IL-10+ cells on CD19+PLT+ or PLT- and (b) percentage of CD19+PLT+ or PLT-IL-10+ according to the percentage of CD19+ producing IL-10 from HD (n = 6) and SLE (n = 9). (c) Correlation of plasma levels of sCD40L and IL-10 of 19 SLE patients. Correlation of plasma levels of IL-10 and (d) CD19+, (e) CD4+, and (f) CD8+PLT+ of 19 SLE patients. Two samples were not included because of the lack of plasma. Data are presented as the mean. Statistical analysis was the Wilcoxon test for paired samples and Mann-Whitney test for unpaired samples. Pearson’s correlation was performed for correlation analysis. ∗ p < 0 05 and ∗∗ p < 0 01. observed in SLE patients with positive anti-dsDNA and lymphocytes, from patients with inflammatory bowel dis- hematuria, a decreased complement C3, and an increased eases are the main source of the elevated sCD40L levels albumin/creatinine ratio. Our results suggest that the binding [41]. In addition, we also found in in vitro cocultures that of platelets to lymphocytes could play a role in SLE disease sCD40L increased proportionally to the platelet-PBMC ratio through the modification of B cell function. [20]. An increase in platelet activation markers such as Joseph et al. described an increase in lymphocyte-platelet thromboxane [42], soluble and surface P-selectin and complexes and higher platelet activation in SLE patients [35]. CD40L [43, 44], and PMPs [45] in the blood of SLE patients However, these authors were unable to discriminate between has been observed in previous studies. Since we found that different populations of lymphocytes. We have found that platelet activation was associated with platelet binding to more B and T lymphocytes from SLE patients had bound lymphocytes in SLE and other authors found that platelet platelets compared with HD and that these levels correlated activation is directly associated with SLE disease activity with plasma sCD40L, a possible indirect measure of platelet [28, 29], it is likely that platelet binding modifies lymphocyte activation [30, 31]. We have also found that T lymphocytes function and, consequently, plays a role in SLE. We and had more bound platelets than B lymphocytes in SLE patients others have shown that platelet bound preferably to memory suggesting a different interaction between platelets and T and T lymphocytes [19, 46]. Therefore, the described expansion B cells of SLE patients. Further experiments will reveal of memory and effector T lymphocytes in SLE patients [47] whether this increase is due to a particular T cell subset or a could explain the increased binding of platelets to SLE than general increase of T cells with bound platelets in SLE. On to HD lymphocytes. Several pairs of molecules, such as the other hand, we did not analyze the activation state of free PSGL-1-P-selectin, have been shown to be involved in the and bound platelets in these patients. It has been shown that lymphocyte-platelet complex formation in HD. However, the increased levels of plasma sCD40L indirectly indicate an we did not find any correlation between PSGL-1 and the increased platelet activation. However, CD40L is overex- percentage of platelet-bound lymphocytes in other inflam- pressed on SLE T cells [28, 32, 33] and this molecule can be matory diseases (unpublished work). According to some shed from the membrane of T lymphocytes [34], contribut- reports, a modification of PSGL-1, the alpha3 fucosylation ing to the soluble pool of sCD40L. On the other hand, using of the O-glycans, is crucial for P-selectin binding and it is in vitro cultures, Danese et al. showed that platelets, but not T restricted to polarized T cells [48, 49]. Therefore, it is likely 8 Mediators of Inflammation Anti-dsDNA Haematuria CD19+ CD4+ CD8+ CD19+ CD4+ CD8+ 15 ⁎⁎⁎ ⁎⁎ ⁎ 15 ⁎ ⁎ ⁎ 10 10 % PLT+ % PLT+ 5 5 0 0 Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative Positive Negative (a) (b) Complement C3 (mg/dl) Albumin/creatinine (mg/mmol) CD19+ CD4+ CD8+ CD19+ CD4+ CD8+ 15 ⁎ 0.09 0.09 15 ⁎ ⁎ ⁎ 10 10 % PLT+ % PLT+ 5 5 0 0 85 − 193 85 − 193 85 − 193 > 2.5 ≤ 2.5 > 2.5 ≤ 2.5 > 2.5 ≤ 2.5 < 85 < 85 < 85 (c) (d) Figure 5: Percentage of lymphocytes with bound platelets in SLE patients according to clinical features. Whole blood from SLE patients (n = 21) was incubated with anti-CD4-PECy7, CD5-PE, CD19-PEDy647, and CD41a-FITC mAbs for flow cytometry analysis. Dot plots of percentages of CD19+ B lymphocytes and CD4+ and CD8+ T lymphocytes with bound platelets in SLE patients segregated according to (a) positive or negative anti-dsDNA, (b) positive or negative hematuria, (c) <85 or 85-193 mg/dl of C3, and (d) >2.5 or ≤2.5 mg/mmol of albumin/creatinine are shown. Data are presented as the mean. Statistical analysis was performed using the Mann-Whitney test. ∗ p < 0 05, ∗∗ p < 0 01, and ∗∗∗ p < 0 001. that the analysis of fucosylated PSGL-1 could be more CD40L on the platelet surface, the binding of platelets to informative to understand the binding of platelets through B lymphocytes [23] through CD40L-CD40 can induce P-selectin to SLE lymphocytes. In a different autoimmune the isotype switching. Accordingly, we found that B lym- context, we found that RA patients with the highest percent- phocytes with bound platelets from SLE patients had ages of CD4+ cells with bound platelets had the lowest levels higher membrane levels of IgG and IgA than B lympho- of IFNγ and IL-17 and the highest levels of plasma IL-10, cytes without bound platelets. An alternative explanation with a less severe disease phenotype [19]. for our findings is that platelets bind preferentially to We found that the percentage of B lymphocytes with IgG+ and IgA+ B lymphocytes. Another intriguing result bound platelets and plasma sCD40L levels correlated with was that B lymphocytes without bound platelets from IgG and IgA levels and negatively correlated with IgM in SLE patients had a higher expression of IgG and IgA than SLE. It is well known that membrane CD40L, like sCD40L, their counterparts from HD. This result could be related is able to induce antibody production and signal B lympho- to the signal provided by the increased levels of sCD40L cytes to switch IgG and IgA [22, 23, 26, 27]. With in the plasma of SLE patients. Mediators of Inflammation 9 Despite the new numbers of preswitched memory, we disease activity and renal manifestations [28, 29]. No dif- showed that more preswitched memory B lymphocytes had ferences in the percentages of B and T lymphocytes with bound platelets in SLE patients than in HD. In addition, bound platelets were found in SLE patients with cutane- more preswitched memory B lymphocytes in SLE patients ous, articular manifestations or when they were segregated had bound platelets than in other B lymphocytes. Our results according to medication, even when we segregated SLE suggest that the preswitched memory stage of B lymphocytes patients taking antiaggregating drugs such as aspirin or favors platelet binding. Two previous observations can hydroxychloroquine. However, this fact was not surprising explain these results. First, we found that B lymphocytes because only clopidogrel, but not aspirin, reduces the forma- with bound platelets had an increased expression of tion of leukocyte-platelet complexes throughout decreasing CD86 and BAFFR compared with B lymphocytes without P-selectin expression on platelets [55]. However, we cannot bound platelets. Second, preswitched memory B lympho- discard that medication of SLE patients could affect lympho- cytes in SLE expressed higher levels of the activation cytes with bound platelets. A study of lymphocyte-platelet markers, CD80, CD95, and CD86, than in HD, as shown by complexes in a large cohort of patients before and after taking Rodriguez-Bayona et al. The affinity of platelets for activated antiaggregating drugs or patients with and without antipho- lymphocytes does not seem to be a mechanism limited to B spholipids antibodies, which is known to be a potent platelet lymphocytes since we, along with other authors, have shown activator, will be required to study the modulation of these that platelets also bind preferentially to activated T lympho- phenomena. We have demonstrated here that, in addition cytes [18, 19], favoring their binding to endothelium to to the secretion of regulatory molecules, platelets can bind migrate [50, 51]. However, with our current experiments, to lymphocytes in SLE. This binding is associated with an we cannot conclude whether the binding of platelet to B lym- altered immune response, and these alterations are related phocytes upregulates the expression of activation markers or to the clinical manifestations of SLE. However, one of our platelets bind preferably to B lymphocytes with an activated limitations is that we have excluded patients under a high phenotype, such as preswitched memory. The addition of dose of glucocorticoids (>10 mg/day) to avoid the influence multiple activation markers for flow cytometry analysis will of this treatment on our results. This exclusion could have also reveal the full activated phenotype of the different biased the cohort to a less severe SLE, and it could explain subsets of B lymphocytes with bound platelets. the low frequency of patients with anti-dsDNA antibodies We showed that B lymphocytes with bound platelets had compared with other published cohorts. Another limitation an increased percentage of IL-10-producing cells compared is that lymphocyte-platelet complexes were analyzed at a with B lymphocytes without bound platelets in SLE patients one-time point in each patient. Further studies analyzing and HD. In addition, the plasma levels of IL-10 in SLE the same patient in a flare and in remission will be required patients correlated with sCD40L plasma levels and the to validate the potential of lymphocyte-platelet complexes percentage of CD19+PLT+, CD4+PLT+, and CD8+PLT+. as a tool to follow up SLE activity. With our current IL-10 has been shown to play a positive role in B lymphocyte approach, we cannot discriminate if the increased percentage survival, proliferation, switching, and autoantibody pro- of B and T lymphocytes with bound platelets is involved in duction [9], contributing to SLE pathology. Accordingly, the development of SLE or if it is a consequence of the plasma IL-10 levels in SLE patients are increased and corre- disease. Nevertheless, our findings suggest that controlling late with SLEDAI and the production of anti-dsDNA anti- this binding is beneficial for the therapeutic regulation of bodies [10]. In addition, increased IL-10 production was autoimmunity. observed in lupus nephritis patients [52]. Our results suggest that increased platelet activation and, consequently, the 5. Conclusions higher number of B lymphocytes with bound platelets could induce the increased IL-10 production of B cells with a detri- Data in the current study show that SLE patients had a higher mental role in the SLE course. However, with our current percentage of circulating lymphocytes with bound platelets experiments, we cannot conclude whether platelets bind to than HD. This elevated percentage was associated with the B lymphocytes producing IL-10 or whether the binding of plasmatic levels of Igs and IL-10 and sCD40L and B cell platelets to B lymphocytes increases the IL-10 production. abnormalities described in SLE. In addition, SLE patients The highest percentages of B and T lymphocytes with with suggestive renal manifestation and active disease had bound platelets were found in those SLE patients with active higher levels of lymphocytes with bound platelets. Taken all disease and those with renal manifestations. Further studies together, our findings suggest that the determination of the with a large cohort of patients and consistent data relating levels of circulating lymphocytes with bound platelets in to the involvement of different organs will be required to find SLE may prove to be a useful tool to follow up the activity associations between platelet bound and lymphocytes with of SLE disease and its renal manifestation. Moreover, different organ manifestations. Since platelet binding is a controlling this binding may prove to be beneficial for the consequence of platelet activation, the activation of platelets therapeutic regulation of autoimmunity. must be associated with the development of lupus nephritis. In experimental models, targeting platelets markedly reduced Abbreviations mortality, proteinuria, kidney histological score, and the levels of anti-DNA antibodies [53, 54]. In SLE patients, plate- CD: Cluster differentiation let activation, measured as sCD40L, is directly related to dsDNA: Double-stranded DNA 10 Mediators of Inflammation HD: Healthy donors [5] M. Bijl, G. Horst, P. Limburg, and C. Kallenberg, “Expression IL: Interleukin of costimulatory molecules on peripheral blood lymphocytes Ig: Immunoglobulin of patients with systemic lupus erythematosus,” Annals of the mAbs: Monoclonal antibodies Rheumatic Diseases, vol. 60, no. 5, pp. 523–526, 2001. PBMCs: Peripheral blood mononuclear cells [6] H. Joo, C. Coquery, Y. 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