Melanocortin 2, 3 and 4 Receptor Gene Expressions are Downregulated in CD8+ T Cytotoxic Lymphocytes and CD19+ B Lymphocytes in Rheumatoid Arthritis Responding to TNF-α Inhibition.

Melanocortin signalling in leucocyte subsets elicits anti-inflammatory and immune tolerance inducing effects in animal experimental inflammation. In man, however, the effects of melanocortin signalling in inflammatory conditions have scarcely been examined. We explored the differential reactions of melanocortin 1-5 receptors (MC1-5R) gene expressions in pathogenetic leucocyte subsets in rheumatoid arthritis (RA) to treatment with TNF-α inhibitor adalimumab. Seven patients with active RA donated blood at start and at 3-month treatment. CD4+ T helper (h) lymphocytes (ly), CD8+ T cytotoxic (c) ly, CD19+ B ly and CD14+ monocytes were isolated, using immunomagnetic beads, total RNA extracted and reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed. Fold changes in MC1-5R, Th1-, inflammatory- and regulatory cytokine gene expressions were assessed for correlation. Six patients responded to adalimumab treatment, while one patient was non-responder. In all lymphocyte subtypes, MC1-5R gene expressions decreased in responders and increased in the non-responder. In responders, decrease in MC2R, MC3R and MC4R gene expressions in CD8+ Tc and CD19+ B ly was significant. Fold change in MC1-5R and IFNγ gene expressions correlated significantly in CD8+ Tc ly, while fold change in MC1R, MC3R and MC5R and IL-1ß gene expressions correlated significantly in CD4+ Th ly. Our results show regulation of MC2R, MC3R and MC4R gene expressions in CD8+ Tc ly and CD19+ B ly. The correlations between fold change in different MCRs and disease driving cytokine gene expressions in CD8+ Tc ly and CD4+ Th ly point at a central immune modulating function of the melanocortin system in RA.

Resistin Gene Expression is Downregulated in CD4(+) T Helper Lymphocytes and CD14(+) Monocytes in Rheumatoid Arthritis Responding to TNF-α Inhibition.

Rheumatoid arthritis (RA) is caused by complex interactions between immune cells and sustained by Th1 response cytokines. Resistin [resistance to insulin; (RETN)] is an inflammatory cytokine, first discovered in murine adipocytes. In man, RETN is mainly secreted by monocytes. The distinct role of RETN in the immune reaction is uncertain; however, RETN has pro-inflammatory, pro-fibrotic and possibly tolerogenic properties. The aim was to assess the reaction of RETN gene expression to TNF-α inhibition (I) in pathogenetic immune cell subsets in RA, in the context of Th1, inflammatory and regulatory cytokine gene expressions. Accordingly, we measured RETN, IFN-γ, TNF-ß, IL-1ß, TNF-α, TGF-ß and IL-10 gene expressions in CD14(+) monocytes, CD4(+) T helper (Th) lymphocytes (ly), CD8(+) T cytotoxic (Tc) ly and CD19(+) B ly in active RA before and 3 months after start of TNF-αI. Leucocyte subsets were separated by specific monoclonal antibody-covered beads, RNA extracted and levels of RETN, Th1 response, inflammatory and regulatory cytokine mRNAs measured by quantitative reverse transcription-polymerase chain reaction technique. We found that TNF-αI caused a significant downregulation of RETN gene expression in CD14(+) monocytes and CD4(+) Th ly and was unchanged in CD8(+) Tc ly and CD19(+) B ly. Both in active RA and during TNF-αI, RETN mRNA levels were significantly higher in CD14(+) monocytes than in all other examined cell types. In monocytes, fold change in RETN and TGF-ß gene expressions upon TNF-αI correlated significantly. Our findings indicate that RETN has pro-inflammatory as well as proresolving roles in active RA.

Dermal melanocortin receptor rebound in diffuse systemic sclerosis after anti-TGFß1 antibody therapy.

Disturbed transforming growth factor beta (TGFß) signalling leads to enhanced synthesis of extracellular matrix (ECM), which is manifested as systemic sclerosis (SSc), but this may be attenuated by the melanocortin system. Here, we report of rebound reaction in the gene expression of melanocortin receptor (MCR) subtypes and of the precursor of these receptors' ligands, the pro-opio-melanocortin protein (POMC), in the acute skin lesion of diffuse systemic sclerosis (dSSc) after treatment with a recombinant human anti-TGFß1 antibody. Biopsies, taken from the leading edge of the skin lesion, before and after treatment of a patient with recent onset dSSc, were examined. Before treatment, increased levels of TGFß mRNA and suppressed levels of POMC mRNA and MCR subtypes MC(1-3, 5) R mRNAs were seen in the lesion, compared with healthy controls. After treatment, there was a rebound expression of POMC, MC(2, 3, 5) R mRNAs. As the melanocortin system regulates collagen and melanin production, our findings add a new understanding to the pathogenetic mechanisms involved in the acute skin lesion of dSSc, which is characterized by enhanced ECM formation and changes in skin pigmentation.

Cytokine mRNA profile of alveolar T lymphocytes and macrophages in patients with systemic sclerosis suggests a local Tr1 response.

The development of an autoimmune disease like systemic sclerosis (SSc) is suspected to be driven by an activated T lymphocyte subset, expressing a cytokine profile specific to the disease. To further characterize the type of immune reaction in SSc, we searched for a broad panel of cytokine messenger ribonucleic acids (mRNAs) in T lymphocytes and monocytes/macrophages from paired samples of bronchoalveolar lavage fluid and peripheral blood in 18 patients and 16 age- and sex-matched controls. RNA from CD3(+) T lymphocytes and CD14(+) monocytes/macrophages was examined by means of the reverse transcriptase polymerase chain reaction. SSc alveolar T lymphocytes expressed a cytokine profile suggestive of a mixed Th1/Th2 reaction, showing an increased frequency of mRNA for interleukin (IL)-10, IL-6 and interferon (IFN)γ, while IL-1ß, IFNγ and tumour necrosis factor ß were expressed in blood T lymphocytes in a higher percentage of patients with SSc than controls. SSc alveolar T cells expressed IL-10 mRNA more often than peripheral T cells, a phenomenon not found in controls and which may point at local IL-10 activation/response in SSc lung. Transforming growth factor ß mRNA was present in all alveolar as well as peripheral blood T cell samples in patients and controls. The cytokine mRNA profile in SSc with interstitial lung disease (ILD) was similar to the profile found in SSc without ILD. Our findings point at a mixed Th1/Th2 reaction in SSc and may indicate regulatory T 1 cell activation/response in the lungs of patients with SSc.

Quantitative measurement of the levels of melanocortin receptor subtype 1, 2, 3 and 5 and pro-opio-melanocortin peptide gene expression in subsets of human peripheral blood leucocytes.

Levels of the melanocortin receptor (MCR) 1, 2, 3 and 5 subtypes and pro-opio-melanocortin (POMC) protein mRNA were measured by the real-time quantitative reverse transcriptase polymerase chain reaction method in CD4+ T helper (Th) cells, CD8+ T cytotoxic cells, CD19+ B cells, CD56+ natural killer (NK) cells, CD14+ monocytes and CD15+ granulocytes from healthy donors. We found high levels of all of the MC1, 2, 3 and 5R subtype mRNA in Th cells and moderate levels in NK cells, monocytes and granulocytes. POMC peptide mRNA was found in all examined leucocyte subsets, but only low levels were present in granulocytes. Our findings suggest a co-ordinating role for MCR subtypes and their naturally occurring ligands in the co-operation between innate and adaptive immunity. Moreover, our findings are compatible with earlier finding of MCR-mediated tolerance induction in Th cells.

Cytoplasmic microvesicular form of Fas ligand in human early placenta: switching the tissue immune privilege hypothesis from cellular to vesicular level.

The local immune privilege of the fetus is created by the placenta. Fas ligand (FasL) expression in trophoblast has been implied as one of the mechanisms of fetal tolerance. However, the expression of membranal FasL by trophoblast has failed to explain this role of FasL. Two objections can be raised: (1) there have been contradictions considering which trophoblast cells, syncytiotrophoblast (ST) or cytotrophoblast, express FasL; (2) in vivo and in vitro studies have shown that the membranal form of FasL evokes inflammatory response and thus may promote fetal rejection. Using different assays and the FasL-specific antibody G247-4 we demonstrate beyond doubt that in vivo, (1) FasL is produced by and stored in the first trimester human ST only and (2) the human ST lacks surface membranal FasL. Instead, FasL, loaded in microvesicles, is stored in cytoplasmic granules. These results complement the recent in vitro studies of the microvesicular form of FasL secretion by cultured trophoblast cells, and suggest that placental FasL is synthesized by villous ST, stored in microvesicular form and secreted as exosomes. Secretion of the exosome-associated form of FasL may be one mechanism by which the placenta promotes a state of immune privilege. Additionally, FasL expression in Hofbauer cells is also demonstrated.

MC(1) receptors are constitutively expressed on leucocyte subpopulations with antigen presenting and cytotoxic functions.

The expression of melanocortin MC(1) receptors on human peripheral lymphocyte subsets was analysed by flow cytometry using rabbit antibodies selective for the human MC(1) receptor and a panel of monoclonal antibodies against lymphocyte differentiation markers. The MC(1) receptor was found to be constitutively expressed on monocytes/macrophages, B-lymphocytes, natural killer (NK) cells and a subset of cytotoxic T-cells. Interestingly T-helper cells appeared to be essentially devoid of MC(1) receptors. The results were confirmed by RT-PCR which indicated strong expression of MC(1) receptor mRNA in CD14(+), CD19(+) and CD56(+) cells. However, only a faint RT-PCR signal was seen in CD3(+) cells, in line with the immuno-staining results that indicated that only part of the CD3(+) cells (i.e. some of the CD8(+) cells) expressed the MC(1) receptor. The MC(1) receptors' constitutive expression on immune cells with antigen-presenting and cytotoxic functions implies important roles for the melanocortic system in the modulation of immune responses.

Lipids are a constitutive component of cytolytic granules.

Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.

gammadelta T cells of human early pregnancy decidua: evidence for cytotoxic potency.

The immune compromise in decidua allows a semiallogeneic fetus to survive without impairing the ability of the maternal immune system to fight infections. Cytotoxic mechanisms are likely to be important in this compromise. Using RT-PCR, immunoflow cytometry and immunoelectron microscopy, the cytotoxic potential of isolated human decidual gammadelta T cells was studied. mRNA for perforin (Pf), granzymes A and B, granulysin and Fas ligand (FasL) was simultaneously expressed in decidual gammadelta T cells. Pf and FasL were not expressed on the cell surface. However, the cells constitutively synthesized Pf and stored it in cytolytic granules. Within the granules Pf mainly resided in the granule core formed by Pf-containing microvesicles. Ultrastructurally, three groups of Pf-containing granules were distinguished. They probably represent different stages of granule maturation in a process where Pf-containing microvesicles first attach to the core cortex and then are translocated across the cortex into the core. Presynthesized FasL was also stored in the core and microvesicles of the cytolytic granules. Upon degranulation by ionomycin/Ca(2+) treatment, FasL was rapidly translocated to the cell surface, demonstrating that its surface expression was not controlled by de novo biosynthesis. Thus decidual gammadelta T cells appear to perform Pf- and FasL-mediated cytotoxicity utilizing a common secretory mechanism based on cytolytic granule exocytosis. The first cytochemical visualization of lipids in the cytolytic granules is provided. These intragranular lipids probably wrap up the core and participate in packaging of the cytotoxic proteins as well as in the killing process. An ultrastructural model of a cytolytic granule is presented.

Gamma delta T cells of human early pregnancy decidua: evidence for local proliferation, phenotypic heterogeneity, and extrathymic differentiation.

The uterine mucosa in pregnancy, the decidua, allows placenta formation and survival of the fetus despite the fact that it is semiallogeneic. Decidua contains large numbers of lymphocytes, of which CD56+ cells dominate, followed by T cells expressing either alpha beta or gamma delta TCR. We have investigated the developmental relationship between the CD56- and TCR gamma delta-expressing cells in early pregnancy decidua using dual labeling immunoelectron microscopy, immunoflow cytometry, and cell fractionation. Lymphocyte subpopulations were, in addition, analyzed for expression of the cytokine receptor for IL-7 and c-kit and for mRNA expression of recombinase-activating genes 1 and 2. Four different cell populations could be distinguished: CD56+bright, CD56+dim/TCR gamma delta+low, CD56+dim/TCR gamma delta+high, and TCR gamma delta+low. Recombinase-activating genes 1 and 2 were expressed in the CD56+bright cells and to a limited degree in CD56+dim/TCR gamma delta+low cells. c-kit was preferentially expressed on the CD56+bright cells, while IL-7R was preferentially expressed on CD56+dim/TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells. The CD56+dim TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells displayed the characteristic morphology of large granular lymphocytes, while single positive TCR gamma delta+low cells were usually smaller and did not contain cytoplasmic granules. The gamma delta 1 gene segment was almost exclusively used in the TCR. Gamma delta T cells in mitosis were seen. We suggest that human early pregnancy decidua is a transient site for extrathymic maturation and that the progenitors of TCR gamma delta+ cells are bone marrow-derived immature cells expressing the CD56 (neural cell adhesion molecule) homing receptor.