Selective Loss of Chemokine Receptor Expression on Leukocytes after Cell Isolation

Chemokine receptors are distinctively exposed on cells to characterize their migration pattern. However, little is known about factors that may regulate their expression. To determine the optimal conditions for an accurate analysis of chemokine receptors, we compared the expression of CCR2, CCR4, CCR5, CCR6, CXCR3 and CXCR4 on different leukocyte subsets using whole blood (WB) plus erythrocyte lysis and density gradient isolation (Ficoll). Most WB monocytes were CCR2+ (93.5±2.9%) whereas 32.8±6.0% of monocytes from Ficoll-PBMC expressed CCR2 (p<0.001). Significant reductions of CCR6 and CXCR3 on monocytes were also observed after Ficoll isolation (WB: 46.4±7.5% and 57.1±5.5%; Ficoll: 29.5±2.2% and 5.4±4.3% respectively) (p<0.01). Although comparable percentages of WB and Ficoll-PBMC monocytes expressed CCR4, CCR5 and CXCR4, Ficoll isolation significantly reduced the levels of CXCR4 (WB: MFI 5±0.4 and Ficoll: MFI 3.3±0.1) (p<0.05). Similarly to monocytes, CCR2, CXCR3 and CXCR4 were also reduced on lymphocytes. In addition, Ficoll isolation significantly reduced the percentage of CCR4 positive lymphocytes (WB: 90.2±4.5% and Ficoll: 55±4.1%) (p<0.01). The loss of expression of chemokine receptors after isolation of monocytes was not dependent on either the anticoagulant or the density gradient method. It was irreversible and could not be restored by LPS activation or in vitro macrophage differentiation. Experiments tagged with anti-CCR2 antibodies prior to density gradient isolation demonstrated that Ficoll internalized chemokine receptors. The method for cell isolation may alter not only the expression of certain chemokine receptors but also the respective functional migration assay. The final choice to analyze their expression should therefore depend on the receptor to be measured

Increase of Circulating Monocyte–Platelet Conjugates in Rheumatoid Arthritis Responders to IL-6 Blockage

Monocytes; Rheumatoid arthritis; TocilizumabMonocitos; Artritis reumatoide; TocilizumabMonòcits; Artritis reumatoide; TocilizumabPlatelets (PLT) bind to a significant percentage of circulating monocytes and this immunomodulatory interaction is increased in several inflammatory and autoimmune conditions. The therapeutic blockage of IL-6 with Tocilizumab (TCZ) alters PLT and the phenotype and function of monocytes in rheumatoid arthritis (RA). However, the relationship between monocyte–PLT conjugates (CD14+PLT+) and clinical and immunological variables and the regulation of this interaction by IL-6 blockage are still unknown. Here, we compared the presence of monocyte–PLT conjugates (CD14+PLT+) and membrane CD162 expression using flow cytometry, and, by ELISA, the markers of PLT activation (sCD62P and sCD40L) in healthy donors (HD) and patients with long-standing RA before TCZ (baseline). We found higher percentages and absolute counts of CD14+PLT+, and higher plasmatic levels of sCD62P and sCD40L but lower CD162 expression on monocytes from RA patients than those from HD. Additionally, the levels of CD14+PLT+ inversely correlated with inflammatory parameters. Interestingly, 95% of patients with lower percentages of CD14+PLT+ and only 63% of patients with higher percentages of CD14+PLT+ achieved a EULAR-defined response at four weeks (p = 0.036). After TCZ, the percentage of CD14+PLT+ increased in 92% of RA patients who achieved 12 w-remission (p < 0.001). Our results suggest that the binding of PLTs has a modulatory effect, accentuated by the increased binding of PLTs to monocytes in response to the therapeutic blockage of IL-6.This study is supported by Instituto de Salud Carlos III and Fondos FEDER (PI17/00072 and PI20/00184)

Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Levels of chemokine receptor expression on leukocytes from WB and Ficoll isolated PBMC.

<p>Representative experiment with leukocytes from healthy donors <i>(n = 12)</i>. Monocytes were stained with anti-CD14 mAbs and lymphocytes and neutrophils were selected by FS and SS parameters. Leukocytes were also stained with monoclonal antibodies for chemokine receptor CCR2, CCR4, CCR5 CCR6, CXCR3 and CXCR4. Profile of expression was assesed by flow cytometry (isotype control <b>□</b> , chemokine receptor ▪).</p

Migration capacity of monocytes from WB and Ficoll PBMC towards CCL2.

<p>WB diluited 1∶5 in medium (100 µl per well) or Ficoll PBMC (2×10⁶ cells/ml per well) were subjected to a 4 h-chemotaxis assay towards CCL2 or fMLP <i>(n = 5)</i>. The cells that had migrated into the lower chamber were collected and stained with anti-CD14-PEDy647 and analyzed by flow cytometry. Results are expressed as the mean of the number of migrated cells ± SEM.</p

Determination of fate of CCR2 downregulation on monocytes from Ficoll.

<p>CCR2 expression was assessed after Ficoll isolation of untagged and tagged-CCR2 monocytes. Internalization of the complex was examined from one single layer of Z stacks by confocal microscopy. The results of a representative experiment are shown: a) CCR2 expression on monocytes from WB tagged with CCR2 prior to gradient separation; b) CCR2 expression on untagged monocytes after Ficoll isolation and the subsequent staining of CCR2; c) CCR2 expression in CCR2 tagged monocytes after Ficoll isolation, red signal within monocytes represents the CCR2-complex internalization (no aditional anti CCR2-PE was added to the cells after isolation with Ficoll); (isotype control <b>□</b> , chemokine receptor ▪).</p

Chemokine receptor expression on LPS and seven-day cultured monocytes.

<p>The results of a representative experiment are shown. a) Monocytes from WB or monocytes from Ficoll were culture 20 h in medium or LPS (0.01 µg/ml) and stained with anti-CCR2-PE or anti-CXCR3-PE. b) Monocytes from Ficoll were cultured in medium and seven days later, cells were harvested and stained with anti-CCR2-PE, anti-CXCR3-PE and anti-CCR5-PE (isotype control <b>□</b> , chemokine receptor ▪).</p

Chemokine receptor expression on leukocytes from peripheral blood.

<p>MFI: Mean Fluorescence Intensity.</p>*<p>p<0.05,</p>**<p>p<0.01,</p>***<p>p<0.001.</p

Effect of anticoagulant and the cell separation method on monocyte chemokine receptors expression.

<p>WB, Ficoll and Percoll isolated PBMC from healthy donors (<i>n = 3</i>) were collected in Vacutainer tubs containing sodium heparine or EDTA. Monocytes were CD14+ gated and CCR2 (clone 48607), CXCR3 and CXCR4 expression were determined. The results of a representative cytometric analysis were shown <i>(n = 5)</i> (isotype control <b>□</b> , chemokine receptor ▪).</p

Rituximab-induced interleukin-15 reduction associated with clinical improvement in rheumatoid arthritis

More lively rear facade with varied rhythm, and yellow brick banding, detail, end block which would have store on ground floor; Oud was affiliated with the De Stijl group, and began to collaborate with artists on housing projects. Oud was appointed in 1918 municipal architect of Rotterdam, a position he held until 1933, and which allowed him the chance to put his theories about mass housing into practice: he was particularly concerned with strip building. Working with the "strip" row house model in the Hook of Holland, he added curved shop windows at the end of the rows, so emphasizing further the horizontality of the solid balcony parapets; these became widely published images upon the project’s completion in 1927. The balconies divide lower and upper floor apartment units, but Oud treats the facades not as an accumulation of individual dwellings but as an architectural whole in which the street elevation functions as part of the urban fabric. In contrast the rear facade is more lively. White facades, yellow brick, blue doors and railings and red lampposts are a De Stijl influence, although he broke with the movement after this building. Source: Grove Art Online; http://www.oxfordartonline.com/ (accessed 8/19/2015