Carlos Zamora

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.

Functional consequences of CD36 downregulation by TLR signals

TLR recognition activates the secretion of pro- and anti-inflammatory cytokines and it also modulates the expression of crucial molecules involved in phagocytosis and antimicrobial activity. Scavenger receptors can act as TLR co-receptors or facilitate antigen loading. However, it remains unknown whether TLR can modulate the expression of these scavenger receptors. We stimulated human peripheral blood mononuclear cells (PBMC) with TLR2 (Pam3CSK4 and FSL1) and TLR4 ligand lipopolysaccharide (LPS) and then analyzed CD36 expression on different monocyte subpopulations by flow cytometry. TLR2 and TLR4 ligands can downregulate CD36 on the surface of monocytes, guiding the protein to intracellular compartments. Even though TLR-activation induced TNFα, IL-10 and IL-6 production, only recombinant TNFα was able to downregulate CD36. Neutralizing anti-TNFα antibodies showed that the Pam3CSK4 and FSL1-induced downregulation was partially mediated by TNFα but not by IL-6 or IL-10. However, LPS-induced downregulation could have also been caused by direct TLR4 targeting and signaling, and/or mediated by other unknown factors. CD36 downregulation reduced the capability of monocytes to phagocyte apoptotic neutrophils. In conclusion, modulation of scavenger receptor expression by TLR targeting on monocytes has functional consequences. Characterization this complex regulation may help us to understand this innate response and develop specific therapeutic drugs for each mechanism.

Functional consequences of platelet binding to T lymphocytes in inflammation

Expression of the scavenger receptor CD36 on lymphocytes is intriguing. We observed that a minor subpopulation of lymphocytes expressed CD36 on the cell surface. We investigated the source of CD36 and also the proliferation and cytokine production of these CD36+ CD4+ lymphocytes. Flow cytometry analysis and immunofluorescence microscopy showed that CD36+ platelets were responsible for CD36 detection on lymphocytes. CD36 was then used as a tool to characterize lymphocytes with bound platelets. Activation‐induced proliferation was lower in CD4+ lymphocytes with bound platelets than lymphocytes without bound platelets. IL‐17 and IFN‐γ production was also reduced in lymphocytes with bound platelets. We then studied the presence of CD36+ CD4+ lymphocytes in RA patients. We observed that the percentage of CD4+ lymphocytes with bound platelets was higher on RA patients than in healthy donors. RA patients with higher titers of anti‐CCP, RF levels, and cardiovascular risk index presented a lower percentage of CD4+ lymphocytes with bound platelets. These patients also had higher IL‐17 and IFN‐γ production. These results suggest that platelet‐binding modifies lymphocyte function. This binding could be a regulatory mechanism in RA that confers a less severe phenotype.

IL-6 blockade reverses the abnormal STAT activation of peripheral blood leukocytes from rheumatoid arthritis patients.

Considering the interplay of multiple STATs in response to cytokines, we investigated how IL-6 and its blocking affect STAT signaling in rheumatoid arthritis (RA). Leukocytes obtained from RA patients before and after tocilizumab treatment and healthy donors (HDs) were cytokine-stimulated and STAT phosphorylation was analyzed by cytometry. RA patients had significantly fewer pSTAT1+, pSTAT3+, and pSTAT6+ monocytes and pSTAT5+ lymphocytes than HDs. After 24weeks of treatment, percentages of IFNγ-induced pSTAT1+ and IL-10-induced pSTAT3+ monocytes in RA patients increased, reaching levels comparable to HDs. pSTAT1+ and pSTAT3+ cells correlated inversely with RA disease activity index and levels of pSTAT+ cells at baseline were higher in patients with good EULAR response to tocilizumab. IFNγ-induced pSTAT1+ cells correlated inversely with memory T cells and anti-CCP levels. IL-10-induced pSTAT3+ cells correlated with Treg/Teff ratio. Our findings suggest that IL-6 blocking reduces the inflammatory mechanisms through the correction of STAT1 and STAT3 activation status.

Binding of Platelets to Lymphocytes: A Potential Anti-Inflammatory Therapy in Rheumatoid Arthritis

Soluble factors released from platelets can modulate the immune response of leukocytes. We and others have recently found that T lymphocytes with bound platelets have reduced proliferation and IFN-γ and IL-17 production. Thus, we speculate that if we induce the binding of platelets to lymphocytes, we will be able to regulate the inflammatory response. When we cocultured platelets with lymphocytes at different ratios, we were able to increase the percentage of lymphocytes with bound platelets. The coculture of platelets with lymphocytes in the presence of stimulation decreased the production of IFN-γ and TNF-α, T cell proliferation, and the expression of CD25, PD-L1, and SLAM. However, this coculture increased CD39 expression. All of these effects were dependent on the dose of platelets and operated indistinctly with platelets from different healthy donors. When platelets were cocultured in the same compartment with lymphocytes, we observed less IFN-γ and TNF-α production and T lymphocyte proliferation than in cultures with platelets separated from lymphocytes by a 0.4-μm pore size filter. The binding of platelets to lymphocytes was blocked with anti–P-selectin Abs, and when this occurred we observed higher IFN-γ and TNF-α production than in nonblocked conditions. The cocultures of platelets with synovial fluid cells from rheumatoid arthritis patients reduced inflammatory cytokine production and increased IL-10 production. These results suggest that platelet binding to lymphocytes effectively regulates T lymphocyte function. This mechanism could be easily applied to reduce inflammatory responses.

The combination of IL-6 and its soluble receptor is associated with the response of rheumatoid arthritis patients to tocilizumab

BACKGROUND IL-6 contributes significantly to the chronic inflammatory process of rheumatoid arthritis (RA). Tocilizumab, a humanized anti-human IL-6 receptor antibody that blocks the signaling originated by the IL-6/IL-6R complex, is an effective treatment. However, predictors of the response to tocilizumab are still required. We aimed to combine IL-6 and soluble IL-6R (sIL-6R) levels to identify groups of responses. METHODS Heparinized blood and clinical data from 63 RA patients were collected before treatment and after 3 and 6 months. Two-step clustering (SPSS v.18) was used to establish the relationship between IL-6 and sIL-6R. Then, we compared European League Against Rheumatism (EULAR) response criteria with remission achievement in the groups of patients. RESULTS Three statistical significant clusters of RA patients (i.e., g1, g2, and g3) were defined by serum concentrations of IL-6 and sIL-6R at baseline. All groups of RA patients had higher IL-6 and sIL-6R levels than healthy donors. The levels of IL-6 expressed as median (IQR) in g1 patients were 124(90-183)pg/ml, in g2 12.3(4.4-24)pg/ml, and in g3 60.1(30-146)pg/ml (p < 0.001). The levels of sIL-6R expressed as mean ± sd in g1 patients were 250.5 ± 72ng/ml, in g2 269.1 ± 125ng/ml, and in g3 732.7 ± 243ng/ml (p < 0.001). Disease activity score (DAS)28, C-reactive protein, and erythrocyte sedimentation rate were comparable in the three groups at baseline. Disease duration in g3 was the longest (median(IQR) years: g1 = 11(5-15), g2 = 12(8-20), and g3 23(16-26); p = 0.006), with years of disease evolution being correlated with sIL-6R levels (R = 0.417, p < 0.001). Simple and Clinical Disease Activity Index (SDAI and CDAI) decreased significantly in the three groups. However, EULAR response criteria and remission achievement at 6m was different in the three groups (p = 0.03 and 0.04, respectively). In all. 17 out of the 18 patients in g1 had a good or moderate response to tocilizumab. Conversely, the percentage of patients with no response to tocilizumab was higher in g3 than in g1 and g2. We also observed different changing patterns of IL-6 and sIL-6R levels among the three groups. CONCLUSIONS RA patients could be easily stratified prior to therapeutic intervention with two molecules related to the pathway blocked by tocilizumab. G1 patients, who had the best response to tocilizumab, had the highest level of IL-6 and the lowest level of sIL-6R.

Ascitic fluid regulates the local innate immune response of patients with cirrhosis

Ascitic neutrophils from cirrhotic patients with spontaneous bacterial peritonitis (SBP) exhibit an impaired oxidative burst that could facilitate bacterial infection. However, the influence of the cell‐free ascitic fluid of these patients on neutrophil function has not been investigated. To analyze this influence, we determined the ascitic levels of cytokines, resistin, and lactoferrin and their association with neutrophil function, disease severity score, and SBP resolution. We analyzed NETosis induction by microscopy and oxidative burst by the flow cytometry of healthy neutrophils cultured in ascitic fluid from cirrhotic patients with sterile ascites (SA) and with SBP before and after antibiotic treatment. Resistin, IL‐6, IL‐1 receptor antagonist, IL‐1β, and lactoferrin levels were measured in ascitic fluids and supernatants of cultured neutrophils and PBMCs by ELISA. Upon stimulation, healthy neutrophils cultured in SBP ascitic fluid produced lower NETosis and oxidative burst than those cultured in SA. Ascitic resistin levels were negatively correlated with NETosis, oxidative burst, and ascitic glucose levels; and positively correlated with the model for end‐stage liver disease score. After an E. coli or TNF‐α stimulus, neutrophils were the major resistin producers. Resistin indirectly reduced the oxidative burst of neutrophils and directly reduced the inflammatory phenotype of monocytes and TNF‐α production. Bacterial‐induced resistin production can down‐regulate the inflammatory response of macrophages and neutrophil function in ascitic fluid. Consequently, this down‐regulation may jeopardize the elimination of bacteria that translocate to ascitic fluid in patients with cirrhosis.

Bacteria-related Events and the Immunological Response of Onset and Relapse Adult Crohn’s Disease Patients

Background and Aims Crohns disease [CD] is a chronic, systemic inflammatory disease characterised by periods of remission and flare-ups. It has been associated with a disturbed gastrointestinal barrier function, an increase in the transport of luminal contents into the tissue, and lower immune tolerance. Methods Peripheral blood samples were collected from healthy controls and 33 adult active flare-up CD patients. We classified patients as onset or relapse flare-up subjects, according to the days of disease evolution. Plasma levels of lipopolysaccharide-binding protein [LBP], fatty acid-binding proteins [FABP], and antibodies against bacterial lysates, interferons [IFN] and interleukin-6 [IL6] were measured by enzyme-linked immunosorbent assay [ELISA] in each group of patients. Results Onset CD patients had higher plasma levels of LBP [57.32 ± 38.86 vs 30.22 ± 9.80 µg/ml] and IFNα [1.25 ± 0.23 vs 0.95 ± 0.36 log10pg/ml] and lower levels of immunoglobulins G and A [IgG and IgA] antibodies against bacterial lysates than relapse CD patients. We also observed a subgroup of onset patients with the highest levels of LBP. In this subgroup, LBP correlated negatively with C-reactive protein [CRP]. Onset and relapse CD patients had similar levels of FABP6 and FABP2, though LBP and FABP6 correlated positively only in relapse patients. In relapse patients, anti-E coli IgG antibodies correlated positively with systemic IL6 and IFNα levels. Conclusions Our findings suggest that onset and relapse flare-ups in adult CD patients are related to different systemic immune-related bacterial events. Characterising these differences may provide insights into the aetiology of Crohns disease, and would help in the selection of appropriate therapies.

CSF-1 regulates the function of monocytes in Crohn’s disease patients in remission

During the flare-ups of Crohn’s disease (CD) patients, circulating leukocytes actively migrate toward the inflamed sites. During the remission, the lack of symptoms does not necessarily imply immunological remission. To decipher inflammatory mechanisms still operating during CD remission, we compared the expression of chemokine receptors on monocytes from CD and healthy donors (HD), and how these differences could modulate monocyte maturation and cytokine production. Flow cytometry analysis showed a higher expression of CCR5 on monocytes from CD patients than those from HD after 24 h. This CCR5 upregulation was associated with the spontaneous production of CSF-1 and IL-10. The higher expression of CCR5 on CD monocytes increased their migratory pattern in response to CCL5. Signaling through CCR5/CCL5 increased CD163 and HLA-DR expression and diminished TLR4-induced TNF-α and IL-6 secretion during monocyte differentiation. When we analyzed clinical parameters, patients treated with azathioprine had the highest CSF-1 levels and CCR5 expression. Our results suggest that monocytes from CD patients in remission produced high levels of CSF-1 that upregulate CCR5 expression. Consequently, monocytes differentiated in these conditions had a characteristic phenotype and lower production of inflammatory cytokines. The treatment with azathioprine could be responsible for this anti-inflammatory profile of monocytes.