High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals

Introduction  

To investigate whether monosodium urate (MSU) crystals induce the production of CCL2 (monocyte chemoattractant protein-1; MCP-1) in human fibroblast-like synoviocytes (FLS) and whether this mechanism would be affected by high-density lipoproteins (HDL).     

Blockade of T Cell Contact-Activation of Human Monocytes by High-Density Lipoproteins Reveals a New Pattern of Cytokine and Inflammatory Genes

Background

Cellular contact with stimulated T cells is a potent inducer of cytokine production in human monocytes and is likely to play a substantial part in chronic/sterile inflammatory diseases. High-density lipoproteins (HDL) specifically inhibit the production of pro-inflammatory cytokines induced by T cell contact.

Methodology/Principal Findings

To further elucidate the pro-inflammatory functions of cellular contact with stimulated T cells and its inhibition by HDL, we carried out multiplex and microarray analyses. Multiplex analysis of monocyte supernatant revealed that 12 out of 27 cytokines were induced upon contact with stimulated T cells, which cytokines included IL-1Ra, G-CSF, GM-CSF, IFNγ, CCL2, CCL5, TNF, IL-1β, IL-6, IL-8, CCL3, and CCL4, but only the latter six were inhibited by HDL. Microarray analysis showed that 437 out of 54,675 probe sets were enhanced in monocytes activated by contact with stimulated T cells, 164 probe sets (i.e., 38%) being inhibited by HDL. These results were validated by qPCR. Interestingly, the cytokines induced by T cell contact in monocytes comprised IL-1β, IL-6 but not IL-12, suggesting that this mechanism might favor Th17 polarization, which emphasizes the relevance of this mechanism to chronic inflammatory diseases and highlights the contrast with acute inflammatory conditions that usually involve lipopolysaccharides (LPS). In addition, the expression of miR-155 and production of prostaglandin E₂—both involved in inflammatory response—were triggered by T cell contact and inhibited in the presence of HDL.

Conclusions/Significance

These results leave no doubt as to the pro-inflammatory nature of T cell contact-activation of human monocytes and the anti-inflammatory functions of HDL.     

Advantages of Papio anubis for preclinical testing of immunotoxicity of candidate therapeutic antagonist antibodies targeting CD28

Antagonist anti-CD28 antibodies prevent T-cell costimulation and are functionally different from CTLA4Ig since they cannot block CTLA-4 and PDL-1 co-inhibitory signals. They demonstrated preclinical efficacy in suppressing effector T cells while enhancing immunoregulatory mechanisms. Because a severe cytokine release syndrome was observed during the Phase 1 study with the superagonist anti-CD28 TGN1412, development of other anti-CD28 antibodies requires careful preclinical evaluation to exclude any potential immunotoxicity side-effects. The failure to identify immunological toxicity of TGN1412 using macaques led us to investigate more relevant preclinical models. We report here that contrary to macaques, and like in man, all baboon CD4-positive T lymphocytes express CD28 in their effector memory cells compartment, a lymphocyte subtype that is the most prone to releasing cytokines after reactivation. Baboon lymphocytes are able to release pro-inflammatory cytokines in vitro in response to agonist or superagonist anti-CD28 antibodies. Furthermore, we compared the reactivity of human and baboon lymphocytes after transfer into non obese diabetic/severe combined immunodeficiency (NOD/SCID) interleukin-2rγ knockout mice and confirmed that both cell types could release inflammatory cytokines in situ after injection of agonistic anti-CD28 antibodies. In contrast, FR104, a monovalent antagonistic anti-CD28 antibody, did not elicit T cell activation in these assays, even in the presence of anti-drug antibodies. Infusion to baboons also resulted in an absence of cytokine release. In conclusion, the baboon represents a suitable species for preclinical immunotoxicity evaluation of anti-CD28 antibodies because their effector memory T cells do express CD28 and because cytokine release can be assessed in vitro and trans vivo.     

HDL Interfere with the Binding of T Cell Microparticles to Human Monocytes to Inhibit Pro-Inflammatory Cytokine Production

Background

Direct cellular contact with stimulated T cells is a potent mechanism that induces cytokine production in human monocytes in the absence of an infectious agent. This mechanism is likely to be relevant to T cell-mediated inflammatory diseases such as rheumatoid arthritis and multiple sclerosis. Microparticles (MP) generated by stimulated T cells (MP_T) display similar monocyte activating ability to whole T cells, isolated T cell membranes, or solubilized T cell membranes. We previously demonstrated that high-density lipoproteins (HDL) inhibited T cell contact- and MP_T-induced production of IL-1β but not of its natural inhibitor, the secreted form of IL-1 receptor antagonist (sIL-1Ra).

Methodology/Principal Findings

Labeled MP_T were used to assess their interaction with monocytes and T lymphocytes by flow cytometry. Similarly, interactions of labeled HDL with monocytes and MP_T were assessed by flow cytometry. In parallel, the MP_T-induction of IL-1β and sIL-1Ra production in human monocytes and the effect of HDL were assessed in cell cultures. The results show that MP_T, but not MP generated by activated endothelial cells, bond monocytes to trigger cytokine production. MP_T did not bind T cells. The inhibition of IL-1β production by HDL correlated with the inhibition of MP_T binding to monocytes. HDL interacted with MP_T rather than with monocytes suggesting that they bound the activating factor(s) of T cell surface. Furthermore, prototypical pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, CCL3 and CCL4 displayed a pattern of production induced by MP_T and inhibition by HDL similar to IL-1β, whereas the production of CCL2, like that of sIL-1Ra, was not inhibited by HDL.

Conclusions/Significance

HDL inhibit both MP_T binding to monocytes and the MP_T-induced production of some but not all cytokines, shedding new light on the mechanism by which HDL display their anti-inflammatory functions.