Endogenous HLA-DR-restricted presentation of the cartilage antigens human cartilage gp-39 and melanoma inhibitory activity in the inflamed rheumatoid joint.

OBJECTIVE: The cartilage proteins melanoma inhibitory activity (MIA) and human cartilage gp-39 (HC gp-39) are candidate autoantigens in rheumatoid arthritis (RA). The present study was undertaken to investigate the endogenous HLA-DR4-restricted presentation of these self proteins, in order to seek in vivo evidence in support of their potential immunologic role. METHODS: MIA and HC gp-39 were assessed in synovial fluid (SF) by enzyme-linked immunosorbent assay and in synovial tissue (ST) by immunohistochemistry. Presentation by SF cells was investigated using specific, HLA-DR-restricted T cell hybridomas. RESULTS: MIA and HC gp-39 were detected in RA SF and ST, as well as in specimens from patients with other forms of arthritis. When HC gp-39-specific and MIA-specific HLA-DR4-restricted T cell hybridomas raised in HLA-DR4-transgenic mice were incubated with RA SF cells as antigen-presenting cells in the presence of HC gp-39 or MIA peptides, the corresponding T cell hybridomas showed strong responses, which were blocked by anti-HLA-DR antibodies. Weaker but qualitatively similar responses were observed with exogenous protein, indicating uptake and processing of these antigens by SF cells. More importantly, without addition of peptide or protein, endogenous presentation of MIA and HC gp-39 was detected in SF cells from 53% and 80% of HLA-DRB1*0401-positive RA patients, respectively. In addition, SF cells from 3 of 10 patients with spondylarthritis exhibited endogenous HC gp-39 presentation. CONCLUSION: These data indicate that immunodominant epitopes of MIA and HC gp-39 are actively presented in an HLA-DR-restricted manner in the inflamed RA joint. The question remains as to whether this leads to activation of autoreactive T cells, which could play a role in either the immunopathology or the immunomodulation of arthritis.

Hormonal stimulation for IVF treatment positively affects the CD56bright/CD56dim NK cell ratio of the endometrium during the window of implantation.

The effects of hormone stimulation for IVF treatment on endometrial receptivity remain controversial. Since CD56(bright) natural killer (NK) cells in the endometrium positively contribute to implantation and decidualization whereas CD56(dim) NK cells are negatively associated with reproduction, shifts in the balance between those cells will affect receptivity. Therefore, we compared the leukocyte composition in the endometrium of IVF women (n=20) with non-pregnant women (n=18) in a natural cycle, as a parameter for endometrial quality. Biopsies were obtained 7 days after ovulation. Histological dating of the endometrium showed no increased endometrial advancement after IVF treatment as compared to the control group. Flow cytometric analysis of leukocyte subsets showed that hormonal stimulation positively affected the CD56(bright)/CD56(dim) ratio in the endometrium by a relative decrease in the cytotoxic CD56(dim)CD16(+) NK cell numbers. The relative number of T-cells remained unaffected, while the number of non-T and non-NK cells (i.e. B-cells and macrophages) was higher in the IVF group. These effects were restricted to the endometrium and not observed in peripheral blood. Within the CD56(bright) population we could identify a distinct subset of NK cells (CD56(superbright)) that was unique for the endometrium. We conclude that hormonal stimulation for IVF treatment positively affects the CD56(bright)/CD56(dim) ratio of the endometrium during the window of implantation and does not affect endometrial advancement.

Membrane-bound HLA-G activates proliferation and interferon-gamma production by uterine natural killer cells.

The expression of HLA-G by invading trophoblasts suggests a role for this molecule in embryo implantation. Putative targets for HLA-G are the uterine natural killer cells (uNK) that are abundantly present at the time of implantation. Since NK cells are potent producers of a variety of cytokines, interaction with HLA-G may result in the production of cytokines involved in trophoblast differentiation or tissue remodelling. In the present study we investigated the effect of membrane-bound HLA-G (mHLA-G) on the uterine mononuclear cell population (UMC) as a whole and on uNK cells in particular by measuring proliferation and cytokine production [interferon-gamma (IFN-gamma)/vascular endothelial growth factor (VEGF)/leukaemia inhibitory factor (LIF)/interleukin-3 (IL-3)]. Uterine cells were isolated from endometrium of non-pregnant women at the time that the endometrium is thought to be receptive to implantation, and then co-cultured with HLA-class I(-)/HLA-class II(+) 721.221 B-LCL cells transfected with mHLA-G. HLA-G suppressed the alloproliferative response of unfractionated UMC to 721.221 cells. Also, IFN-gamma and IL-3 production was strongly reduced. In contrast, purified uNK cells were stimulated by mHLA-G. Proliferation as well as IFN-gamma production was increased after co-culture with mHLA-G transfected 721.221 cells. HLA-G stimulated VEGF production by UMC as well as purified uNK cells. LIF-levels were below the detection level of our enzyme-linked immunosorbent assay. In conclusion, our data show that mHLA-G stimulates proliferation and cytokine production by NK cells, while down-modulating the response of unfractionated UMC.

Detection of HLA-G by a specific sandwich ELISA using monoclonal antibodies G233 and 56B.

Human leukocyte antigen (HLA)-G, which is mainly expressed at the maternal-fetal interface, may play a role in the immune tolerance of the semi-allogenic fetus by the mother. Functional studies have shown that HLA-G is indeed a potential modulator of different immune responses. Therefore, it is of interest to study the level of expression of soluble HLA-G in several biological fluids derived from women with and without fertility problems. In order to measure soluble HLA-G, a reliable and sensitive HLA-G specific sandwich ELISA is required. Here, we describe such an ELISA in which G233 is used as the coating antibody and 56B as the detecting antibody. In comparison with two other assays, this assay shows highest responses to recombinant HLA-G and native HLA-G in primary trophoblast culture supernatant and high responses to HLA-G in amniotic fluid. No HLA-G in follicular fluid or preimplantation embryo culture supernatant could be detected.