Anti-apolipoprotein A-1 autoantibodies correlate with disease activity in systemic lupus erythematosus.

OBJECTIVES: Apolipoprotein A-1 (ApoA-1) is a protein fraction of the high-density lipoproteins with anti-inflammatory and antioxidant properties that play a major role in reverse cholesterol transport. The presence of anti-ApoA-1 IgG has been reported in SLE to be variably associated with disease activity or cardiovascular events (CVEs). We assessed the clinical performance of anti-ApoA-1 IgG and of antibodies directed against its immunodominant F3L1 peptide (F3L1 IgG) in a well-characterized Swiss SLE cohort study. METHODS: A total of 354 biological samples and interviews from 176 individuals were studied. SLEDAI, clinical characteristics, anamnestic CVEs and therapy details were recorded. Sera were tested for the presence of anti-ApoA-1 IgG, anti-F3L1 IgG, anti-dsDNA IgG and aPL. RESULTS: Anti-ApoA-1 and anti-F3L1 IgG positivity was associated with higher SLEDAI, mostly due to concomitant positivity of dsDNA IgG and low complement. Variations in time of anti-ApoA-1 IgG correlated positively with variations of anti-dsDNA IgG and inversely to variations of C3 levels. No cross-reactivity was found between anti-ApoA-1 and anti-dsDNA IgG. Positivity for anti-Apo-A1 IgG was more frequent in individuals receiving 10 mg/day or more of prednisone. We did not find any significant association between anti-ApoA-1 IgG positivity and CVEs. CONCLUSION: Anti-ApoA-1 and anti-F3L1 IgG in SLE correlate strongly with laboratory markers of activity, particularly with the presence and titre of dsDNA IgG. These results confirm and extend previous findings and support the use of anti-ApoA1 IgG in the clinical setting. Their role in CVEs deserves further investigation.

Blocking interferon γ reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis.

BACKGROUND: Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA. METHODS: Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs). RESULTS: Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries. CONCLUSIONS: Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall.

Ex Vivo Signaling Protein Mapping in T Lymphocytes in the Psoriatic Arthritis Joints.

We assessed signaling protein mapping in total T cells, to analyze the proportions of T regulatory (Treg) and TCD4+ effector (Teff) cell phenotypes, and the respective interleukin 6Rα (IL-6Rα) expression in the inflammatory microenvironment of synovial fluid (SF) of patients with sustained psoriatic arthritis (PsA). Our approach was to measure the IL-6 level in SF using a multiplex bead immunoassay. Reverse-phase protein array was used to assess Janus kinase (JAK) 1 and JAK2, extra-cellular regulated kinase (ERK) 1 and 2, protein kinase Cδ (PKCδ), signal transducer and activator and transcription (STAT) 1, STAT3, and STAT5 phosphoproteins in total T cell lysates from SF of patients with PsA. Frequencies of CD4+IL-17A-F+IL-23+ CD4+ Th cells producing IL-17A and IL-17F (Th17) and CD4+CD25high intracellular forkhead box transcription factor+ (FOXP3+) phenotypes, and the percentage of Treg- and Teff- cells were quantified in SF and matched peripheral blood (PB) of patients with PsA and PB of healthy controls (HC) by flow cytometry. Our results were the following: In PsA SF samples, a coordinate increase of JAK1, ERK1/2, STAT1, STAT3, and STAT5 phosphoproteins was found in total T cells in SF of PsA; where IL-6 levels were higher than in PB from HC. Expanded CD4+IL-17A-F+IL-23+ Th17, CD4+ CD25- Teff- and CD4+CD25(high) FoxP3+Treg subsets, showing similar levels of enhanced IL-6Rδ expression, were confined to PsA joints. In our studies, the transcriptional network profile identified by ex vivo signaling protein mapping in T lymphocytes in PsA joints revealed the complex interplay between IL-1, IL-6, and IL-23 signaling and differentiation of Th17 cells and CD4+Tregs in sustained joint inflammation in PsA.

Changes in biomarkers after therapeutic intervention in temporal arteries cultured in Matrigel: a new model for preclinical studies in giant-cell arteritis.

BACKGROUND: Search for therapeutic targets in giant-cell arteritis (GCA) is hampered by the scarcity of functional systems. We developed a new model consisting of temporal artery culture in tri-dimensional matrix and assessed changes in biomarkers induced by glucocorticoid treatment. METHODS: Temporal artery sections from 28 patients with GCA and 22 controls were cultured in Matrigel for 5 days in the presence or the absence of dexamethasone. Tissue mRNA concentrations of pro-inflammatory mediators and vascular remodelling molecules was assessed by real-time RT-PCR. Soluble molecules were measured in the supernatant fluid by immunoassay. RESULTS: Histopathological features were exquisitely preserved in cultured arteries. mRNA concentrations of pro-inflammatory cytokines (particularly IL-1ß and IFNγ), chemokines (CCL3/MIP-1α, CCL4/MIP-1ß, CCL5/RANTES) and MMP-9 as well as IL-1ß and MMP-9 protein concentrations in the supernatants were significantly higher in cultured arteries from patients compared with control arteries. The culture system itself upregulated expression of cytokines and vascular remodelling factors in control arteries. This minimised differences between patients and controls but underlines the relevance of changes observed. Dexamethasone downregulated pro-inflammatory mediator (IL-1ß, IL-6, TNFα, IFNγ, MMP-9, TIMP-1, CCL3 and CXCL8) mRNAs but did not modify expression of vascular remodelling factors (platelet derived growth factor, MMP-2 and collagens I and III). CONCLUSIONS: Differences in gene expression in temporal arteries from patients and controls are preserved during temporal artery culture in tri-dimensional matrix. Changes in biomarkers elicited by glucocorticoid treatment satisfactorily parallel results obtained in vivo. This may be a suitable model to explore pathogenetic pathways and to perform preclinical studies with new therapeutic agents.

Cytokine levels in human synovial fluid during the different stages of acute gout: role of transforming growth factor ß1 in the resolution phase.

OBJECTIVES: To determine the most relevant parameters in synovial fluid (SF) during the various stages of acute gout. METHODS: SFs from 38 gouty patients were analysed for white blood cell (WBC) count, percentage of polymorphonuclear cells (PMNs) and levels of interleukin 1ß (IL-1ß), IL-6, IL-8, tumour necrosis factorα (TNFα) and transforming growth factor ß1 (TGFß1). Patients were divided into three groups according to the length of time since onset of the attack: phase I (0-48 h), phase II (days 3-4) and phase III (days 5-7). RESULTS: Levels of WBCs were similar in SFs from phases I and II, while phase III showed the lowest WBC count. Percentages of PMNs were raised in all SFs. None of the cytokines analysed differed between phases I and II except for TGFß1, which was higher in phase II. IL-1ß, IL-6 and TNFα were higher in group 1 than in group 3. Levels of all the cytokines assessed, with the exception of TGFß1, were significantly lower in phase III than in phase II IL-1ß, p<0.05; IL-6, p<0.01; IL-8, p<0.001; TNFα, p<0.05).TGFß1 levels were highest in SFs from phase III. CONCLUSION: Cytokine levels in SFs may change depending on the different stages of acute gout, highlighting the role of TGFß1 in the resolution of gout.

Serum IL-6 and IL-21 are associated with markers of B cell activation and structural progression in early rheumatoid arthritis: results from the ESPOIR cohort.

OBJECTIVE: To identify a specific pattern of serum cytokines that correlates with the diagnosis, activity and severity of rheumatoid arthritis (RA) in patients with early RA as well as with the level of serum markers of B cell activation. METHODS: Serum interleukin (IL)-1ß, IL-1 receptor antagonist (IL1-Ra), IL-2, IL-4, IL-6, IL-10, IL-17, IL-21, monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor α and interferon γ levels were measured in the (ESPOIR) Etude et Suivi des POlyarthrites Indifférenciées Récentes early arthritis cohort, which included patients with at least two swollen joints for >6 weeks and <6 months, and no previous corticosteroids or disease-modifying antirheumatic drugs. Serum cytokine levels were compared between patients who met the 1987 American College of Rheumatology criteria for RA (n=578) or had undifferentiated arthritis (UA, n=132) at the 1-year follow-up visit. RESULTS: Serum IL-6 and IL-21 were the only cytokines that discriminated RA from UA on univariate analysis. IL-6 level was associated with RA, whereas erythrocyte sedimentation rate and C-reactive protein were not. Higher proportions of rheumatoid factor and anticyclic citrullinated protein (CCP) positivity, levels of markers of B cell activation, and a higher frequency of rapid radiographic progression were observed in patients with RA with detectable IL-6 or IL-21. Multivariate analysis associated IL-6 and anti-CCP levels with radiographic erosions at enrolment with 1-year radiographic progression. CONCLUSION: Serum IL-6 concentration is greater in RA than in UA. Increase in serum IL-6 and IL-21 levels is associated with markers of B cell activation, and IL-6 is associated with radiographic progression in patients with RA.

Synovial biomarkers in psoriatic arthritis.

OBJECTIVE: To find candidate biomarkers of psoriatic arthritis (PsA). A panel of synovial fluid (SF) and synovial tissue (ST) biomarkers was analyzed in patients with resistant peripheral PsA, in relation to clinical and imaging outcomes of synovitis response following serial intraarticular (IA) etanercept injections (12.5 mg). METHODS: Fourteen PsA patients with resistant knee joint synovitis were treated with 4 IA etanercept injections in a single knee joint, once every 2 weeks. Primary outcome (Thompson's knee index: THOMP) and secondary outcomes were assessed at baseline and end of study: C-reactive protein, Knee Joint Articular Index (KJAI), Health Assessment Questionnaire disability index, maximal synovial thickness (MST) by gray-scale ultrasonography, contrast-enhanced magnetic resonance imaging (C+MRI), ST-cluster differentiation (CD)45+ mononuclear cell, ST-CD31+ vessels, and ST-CD105+ angiogenic endothelial cells, along with levels of SF interleukin 1ß (IL-1ß), IL-1 receptor antagonist (Ra), and IL-6. RESULTS: At the end of the study, clinical and imaging outcomes, ST and SF biological markers were significantly reduced compared to baseline. There was a significant association between IL-6 and either THOMP or KJAI; between either ST-CD31+ or ST-CD105+ or ST-CD45+; between ST and SF biomarkers expression (CD45+ and IL-1ß) and between ST-CD45+ and both KJAI and MRI-MST. Comparing pre- versus post-IA etanercept injection changes (Δ), Δ IL-1ß was significantly correlated with both Δ IL-6 and with Δ IL-1Ra and Δ IL-6 with Δ IL-1Ra. CONCLUSION: The association to disease activity and the changes following IA treatment indicate that ST-CD45+ and ST-CD31+, along with SF-IL-6 and SF-IL-1ß, may represent candidate biomarkers of the knee synovitis response to IA tumor necrosis factor-α blockade.

Activation of the aryl hydrocarbon receptor reveals distinct requirements for IL-22 and IL-17 production by human T helper cells.

Ligands of the aryl hydrocarbon receptor (AHR), a transcription factor mediating the effects of dioxin, favor Th17 differentiation and exacerbate autoimmunity in mice. We investigated how AHR ligands affected human T-cell polarization. We found that the high affinity and stable AHR-ligand dioxin as well as the natural AHR-ligand 6-formylinolo[3,2-b] carbazole induced the downstream AHR-target cytochrome P450A1, and without affecting IFN-gamma, they enhanced IL-22 while simultaneously decreasing IL-17A production by CD4(+) T cells. The specific AHR-inhibitor CH-223191 abolished these effects. Furthermore, blockade of IL-23 and IL-1, important for Th17 expansion, profoundly decreased IL-17A but not IL-22 production. AHR agonists reduced the expression of the Th17 master transcription factor retinoic acid-related orphan receptor C (RORC), without affecting T-bet, GATA-3 and Foxp3. They also decreased the expression of the IL-23 receptor. Importantly, AHR-ligation did not only decrease the number of Th17 cells but also primed naïve CD4(+) T cells to produce IL-22 without IL-17 and IFN-gamma. Furthermore, IL-22 single producers did not express CD161, which distinguished them from the CD161(+) Th17 cells. Hence, our data provide compelling evidence that AHR activation participates in shaping human CD4(+) T-cell polarization favoring the emergence of a distinct subset of IL-22-producing cells that are independent from the Th17 lineage.

Type I Interferons in Systemic Autoimmune Diseases: Distinguishing Between Afferent and Efferent Functions for Precision Medicine and Individualized Treatment.

A sustained increase in type I interferon (IFN-I) may accompany clinical manifestations and disease activity in systemic autoimmune diseases (SADs). Despite the very frequent presence of IFN-I in SADs, clinical manifestations are extremely varied between and within SADs. The present short review will address the following key questions associated with high IFN-I in SADs in the perspective of precision medicine. 1) What are the mechanisms leading to high IFN-I? 2) What are the predisposing conditions favoring high IFN-I production? 3) What is the role of IFN-I in the development of distinct clinical manifestations within SADs? 4) Would therapeutic strategies targeting IFN-I be helpful in controlling or even preventing SADs? In answering these questions, we will underlie areas of incertitude and the intertwined role of autoantibodies, immune complexes, and neutrophils.

Effect of pathogenic crystals on the production of pro- and anti-inflammatory cytokines by different leukocyte populations.

PURPOSE: To evaluate cytokine production in vitro by different types of leukocytes stimulated with monosodium urate (MSU), calcium pyrophosphate (CPP) and basic calcium phosphate (BCP) crystals. MATERIAL AND METHODS: Polymorphonuclear cells (PMN), monocytes and lymphocytes, isolated from healthy volunteer blood, were stimulated for different time periods with increasing MSU, CPP or BCP crystal concentrations. IL-1ß, IL-8, IL-6, CCL2, IL-1Ra and TGFß1 were determined by ELISA. RESULTS: Exposure of PMN to different crystals resulted in a moderate IL-8 and IL-1Ra release. Stimulation of monocytes induced a significant production of all the cytokines evaluated. The highest levels of IL-1ß, IL-6, CCL2 and IL-8 were observed with MSU at 0.5 mg/ml, CPP at 0.01-0.05 mg/ml and BCP at 1 mg/ml after 18-48 h and then decreased. At the same crystal concentrations, IL-1Ra and TGFß1 increased until the end of the experiment. Treatment of lymphocytes with different crystals did not induce cytokine release. CONCLUSION: This study demonstrates that PMN, monocytes and lymphocytes from the same donor respond differently after stimulation with MSU, CPP or BCP crystals, depending on the dose and the time of exposure. Crystals induce a rapid increase of pro-inflammatory cytokines, whereas longer time is required to release high levels of anti-inflammatory cytokines.

Synovial tissue and serum biomarkers of disease activity, therapeutic response and radiographic progression: analysis of a proof-of-concept randomised clinical trial of cytokine blockade.

OBJECTIVES: To evaluate synovial tissue and serum biomarkers of disease activity, therapeutic response and radiographic progression during biological therapy for rheumatoid arthritis (RA). METHODS: Patients with active RA entered a randomised study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 microg/kg twice a week. Arthroscopic synovial tissue biopsies were obtained at baseline and two further time points. Following immunohistochemical staining, selected mediators of RA pathophysiology were quantified using digital image analysis. Selected mediators were also measured in the serum. RESULTS: Twenty-two patients were randomly assigned: 11 received monotherapy and 11 combination therapy. American College of Rheumatology 20, 50 and 70 response rates were 64%, 64% and 46% with combination therapy and 36%, 9% and 0% with monotherapy, respectively. In synovial tissue, T-cell infiltration, vascularity and transforming growth factor beta (TGFbeta) expression demonstrated significant utility as biomarkers of disease activity and therapeutic response. In serum, interleukin 6 (IL-6), matrix metalloproteinase (MMP) 1, MMP-3 and tissue inhibitor of metalloproteinase 1 (TIMP-1) were most useful in this regard. An early decrease in serum levels of TIMP-1 was predictive of the later therapeutic outcome. Pretreatment tissue levels of T-cell infiltration and the growth factors vascular endothelial growth factor/TGFbeta, and serum levels of IL-6, IL-8, MMP-1, TIMP-1, soluble tumour necrosis factor receptor types I and II and IL-18 correlated with radiographic progression. CONCLUSIONS: Synovial tissue analysis identified biomarkers of disease activity, therapeutic response and radiographic progression. Biomarker expression in tissue was independent of the levels measured in the serum.

Therapeutic targets in rheumatoid arthritis: the interleukin-6 receptor.

RA is a chronic, debilitating disease in which articular inflammation and joint destruction are accompanied by systemic manifestations including anaemia, fatigue and osteoporosis. IL-6 is expressed abundantly in the SF of RA patients and is thought to mediate many of the local and systemic effects of this disease. Unlike a number of other cytokines, IL-6 can activate cells through both membrane-bound (IL-6R) and soluble receptors (sIL-6R), thus widening the number of cell types responsive to this cytokine. Indeed, trans-signalling, where IL-6 binds to the sIL-6R, homodimerizes with glycoprotein 130 subunits and induces signal transduction, has been found to play a key role in acute and chronic inflammation. Elevated levels of IL-6 and sIL-6R in the SF of RA patients can increase the risk of joint destruction and, at the joint level, IL-6/sIL-6R can stimulate pannus development through increased VEGF expression and increase bone resorption as a result of osteoclastogenesis. Systemic effects of IL-6, albeit through conventional or trans-signalling, include regulation of acute-phase protein synthesis, as well as hepcidin production and stimulation of the hypothalamo-pituitary-adrenal axis, the latter two actions potentially leading to anaemia and fatigue, respectively. This review aims to provide an insight into the biological effects of IL-6 in RA, examining how IL-6 can induce the articular and systemic effects of this disease.

Prostaglandin E2 synergistically with interleukin-23 favors human Th17 expansion.

Microenvironment molecular cues direct T helper (Th) cell differentiation; however, Th17 fate determination is still imprecisely understood in humans. To assess the role of prostaglandin E(2) (PGE(2)) in Th expansion, we activated peripheral blood mononuclear cells by CD3 cross-linking. In the presence of exogenous PGE(2), peripheral blood mononuclear cells produced higher interleukin-17 (IL-17), C-C chemokine ligand 20 (CCL20)/macrophage inflammatory protein 3alpha (MIP-3alpha), CXC chemokine ligand 8 (CXCL8)/IL-8, and lower interferon-gamma and IL-22 levels than in control cultures. Exogenous PGE(2) and IL-23 synergized in inducing IL-17, whereas indomethacin and IL-23 blockade drastically reduced IL-17 but not interferon-gamma production. Furthermore, IL-1 but not tumor necrosis factor was absolutely required for IL-17 production. PGE(2) doubled the frequency of CD4+ T cells producing IL-17 and within the CD4+ subset enhanced C-C chemokine receptor 6 (CCR6) and CCR4 while decreasing CXC chemokine receptor 3 (CXCR3) expression. Furthermore, in CD4+ T-cell lines, the production of IL-17 segregated with the CCR6+ subset. In the presence of CCR6+ compared with CXCR3+ Th cells, monocytes/macrophages produced much higher levels of matrix metalloproteinase-1, -3, and -9 but similar levels of CXCL10 and IL-1beta. These results identify PGE(2) and IL-23 as participating in the expansion of CD4+ T cells endowed with high IL-17 production capacity, which in turn favors monocyte production of mediators important for host defense and tissue destruction.

Synovial tissue rank ligand expression and radiographic progression in rheumatoid arthritis: observations from a proof-of-concept randomized clinical trial of cytokine blockade.

The objective of the study was to evaluate synovial tissue receptor activator of nuclear factor-κß ligand (RANKL) and osteoprotegerin (OPG) as biomarkers of disease activity, progressive joint damage, and therapeutic response, during cytokine blockade in rheumatoid arthritis (RA). Patients with active RA entered a randomized open-label 12-month study of anakinra 100 mg/day, administered as monotherapy or in combination with pegsunercept 800 µg/kg twice weekly. Arthroscopic synovial tissue biopsies were obtained at baseline, at 4 weeks and at the final time point. Following immunohistochemical staining, RANKL and OPG expression was quantified using digital image analysis. Radiographic damage was evaluated using the van der Heijde modification of the Sharp scoring system. Twenty-two patients were randomized. Baseline expression of RANKL, but not OPG, correlated significantly with baseline CRP levels (r = 0.61, P < 0.01). While a significant reduction in OPG expression following treatment was observed in clinical responders at the final time point (P < 0.05 vs. baseline), RANKL levels did not change, and the RANKL:OPG ratio remained unaltered, even at the highest levels of clinical response. When potential predictors of radiographic outcome were evaluated, baseline RANKL expression correlated with erosive progression at 1 year (r = 0.71, P < 0.01). Distinct, though related, pathophysiologic processes mediate joint inflammation and destruction in RA. Elevated synovial tissue RANKL expression is associated with progressive joint erosion, and may be independent of the clinical response to targeted therapy. The potential therapeutic importance of modulating RANKL in RA is highlighted, if radiographic arrest is to be achieved.

Immunological determinants of clinical outcome in Peruvian patients with tegumentary leishmaniasis treated with pentavalent antimonials.

The mechanisms linking the immune response to cutaneous and mucosal leishmaniasis (CL and ML, respectively) lesions and the response to treatment are incompletely understood. Our aims were to prospectively assess, by quantitative reverse transcription-PCR, the levels of mRNA for gamma interferon, tumor necrosis factor alpha, interleukin-10 (IL-10), IL-4, and IL-13, as well as the presence of T cells (CD2) and macrophages (CD68), in CL and ML lesions and to follow their changes in response to treatment with pentavalent antimonials. The leishmanin skin test (LST) was performed on all CL and ML patients before treatment. The patient population included individuals living in areas of Peru where the disease is endemic, i.e., 129 with CL and 43 with ML. Compared to CL patients, the LST induration size was larger, the levels of all cytokine mRNAs but IL-10 were higher, T-cell mRNA was similar, and macrophage mRNA was lower in ML patients. The proportion of CL patients with an LST induration size of >8 mm was higher among responders to treatment. In CL, the pretreatment levels of cytokine mRNAs did not discriminate between responders and nonresponders; however, treatment was more often accompanied by a reduction in the levels of T-cell and cytokine mRNAs in responders than in nonresponders. Furthermore, the production of cytokines per T cell and macrophage decreased with treatment but IL-10 production remained high in nonresponders. Overall, these findings point to complex relationships among New World Leishmania parasites, skin and mucosal immune responses, and treatment outcome. The persistence of high levels of IL-10 in CL is characteristically associated with a poor response to treatment.

IL-21 promotes survival and maintains a naive phenotype in human CD4+ T lymphocytes.

IL-21 is a key T-cell growth factor (TCGF) involved in innate and adaptive immune response. It contributes to the proliferation of naive, but not memory T lymphocytes. However, the full spectrum of IL-21 activity on T cells remains unclear. Here, we demonstrate that IL-21 primarily maintains the expression of specific naive cell surface markers such as CD45RA, CD27, CD62L and CCR7 on human CD4(+) T lymphocytes and that the expression of CCR7 induces cell migration by means of CCL21 chemoattraction. These effects contrast with those of IL-2 which induced the marked proliferation of CD4(+) T lymphocytes, leading to an activated-memory phenotype. Nevertheless, IL-21 maintained cell cycle activation and expression of proliferation markers, including proliferating cell nuclear antigen and Ki-67, and triggered T-cell proliferation via TCR and co-stimulation pathways. Unlike IL-2, IL-21 decreased the expression of the anti-apoptotic Bcl-2 protein, which correlated with the absence of activation of the phosphatidylinositol 3'-kinase/Akt signaling pathway. Thus, IL-21 is a TCGF whose function is the preservation of a pool of CD4(+) T lymphocytes in a naive phenotype, with a low proliferation rate but with the persistence of cell cycling proteins and cell surface expression of CCR7. These findings strongly suggest that IL-21 plays a part in innate and adaptive immune response owing to homeostasis of T cells and their homing to secondary lymphoid organs.

Stimulated T cells generate microparticles, which mimic cellular contact activation of human monocytes: differential regulation of pro- and anti-inflammatory cytokine production by high-density lipoproteins.

Imbalance in cytokine homeostasis plays an important part in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis and rheumatoid arthritis. We demonstrated that T cells might exert a pathological effect through direct cellular contact with human monocytes/macrophages, inducing a massive up-regulation of the prototypical proinflammatory cytokines IL-1beta and TNF. This mechanism that might be implicated in chronic inflammation is specifically inhibited by high-density lipoproteins (HDL). Like many other stimuli, besides proinflammatory cytokines, the contact-mediated activation of monocytes induces the production of cytokine inhibitors such as the secreted form of the IL-1 receptor antagonist (sIL-1Ra). The present study demonstrates that stimulated T cells generate microparticles (MP) that induce the production of TNF, IL-1beta, and sIL-1Ra in human monocytes; the production of TNF and IL-1beta but not that of sIL-1Ra is inhibited in the presence of HDL. The results were similar when monocytes were stimulated by whole membranes of T cells or soluble extracts of the latter. This suggests that MP carry similar monocyte-activating factors to cells from which they originate. Thus, by releasing MP, T cells might convey surface molecules similar to those involved in the activation of monocytes by cellular contact. By extension, MP might affect the activity of cells, which are usually not in direct contact with T cells at the inflammatory site. Furthermore, this study demonstrates that HDL exert an anti-inflammatory effect in nonseptic activation of human monocytes, not only by inhibiting the production of IL-1beta and TNF but also, by leaving sIL-1Ra production unchanged.

Differential regulation of cytokine production by PI3Kdelta in human monocytes upon acute and chronic inflammatory conditions.

Deregulated production of cytokines, including IL-1beta, IL-6 and TNF plays an important role in chronic inflammation. Relevant to this condition, direct cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes/macrophages. We previously demonstrated that PI3Ks regulate differential production of IL-1beta and its specific inhibitor secreted IL-1 receptor antagonist (sIL-1Ra) by human monocytes. Here we show that in contrast with PI3Kalpha, beta and gamma, PI3Kdelta accounts for most of the PI3K-dependent signaling ruling the production of IL-1beta, IL-6, TNF and sIL-1Ra in monocytes activated by cellular contact with stimulated T cells (mimicked by CHAPS-solubilized membranes of stimulated T cells, CE sHUT) and lipopolysaccharides (LPS); the latter stimuli being relevant to chronic/sterile and acute/infectious inflammation, respectively. Interestingly, PI3Kdelta activity dampened the production of pro-inflammatory cytokines in LPS-activated monocytes, but induced it in CE sHUT-activated cells. In both CE sHUT- and LPS-activated monocytes PI3Kdelta regulated cytokine transcript expression through the phosphorylation/inactivation of glycogen synthase kinase-3beta (GSK3beta). The blockade of GSK3beta displayed inverse effects to those of PI3Kdelta blockade. Thus, by displaying opposite functions in conditions mimicking chronic/sterile and acute/infectious inflammation, i.e., by repressing pro-inflammatory cytokine expression in LPS-activated monocytes but inducing such mediators in T cell contact-activated monocytes, PI3Kdelta represents a potential therapeutic target specific to chronic/sterile inflammatory conditions.

In vivo investigations on anti-fibrotic potential of proteasome inhibition in lung and skin fibrosis.

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

Anti-(apolipoprotein A-1) IgGs are associated with high levels of oxidized low-density lipoprotein in acute coronary syndrome.

ApoA-1 (apolipoprotein A-1) is the main component of HDL (high-density lipoprotein) and stabilizes PON-1 (paraoxonase-1), which prevents lipid peroxidation and oxLDL (oxidized low-density lipoprotein) formation. Autoantibodies against apoA-1 [anti-(apoA-1) IgG] have been found in antiphospholipid syndrome and systemic lupus erythematosous, two diseases with an increased risk of thrombotic events, as well as in ACS (acute coronary syndrome). OxLDL levels are also elevated in these diseases. Whether anti-(apoA-1) IgGs exist in other prothrombotic conditions, such as APE (acute pulmonary embolism) and stroke, has not been studied and their potential association with oxLDL and PON-1 activity is not known. In the present study, we determined prospectively the prevalence of anti-(apoA-1) IgG in patients with ACS (n=127), APE (n=58) and stroke (n=34), and, when present, we tested their association with oxLDL levels. The prevalance of anti-(apoA-1) IgG was 11% in the ACS group, 2% in the control group and 0% in the APE and stroke groups. The ACS group had significantly higher median anti-(apoA-1) IgG titres than the other groups of patients. Patients with ACS positive for anti-(apoA-1) IgG had significantly higher median oxLDL values than those who tested negative (226.5 compared with 47.7 units/l; P<0.00001) and controls. The Spearman ranked test revealed a significant correlation between anti-(apoA-1) IgG titres and serum oxLDL levels (r=0.28, P<0.05). No association was found between PON-1 activity and oxLDL or anti-(apoA-1) IgG levels. In conclusion, anti-(apoA-1) IgG levels are positive in ACS, but not in stroke or APE. In ACS, their presence is associated with higher levels of oxLDL and is directly proportional to the serum concentration of oxLDL. These results emphasize the role of humoral autoimmunity as a mediator of inflammation and coronary atherogenesis.