Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells

Cultures of human thymic epithelial cells (TEC) were tested for the expression of HLA class I (A, B, C) and class II (DR and DC) antigens by indirect immunofluorescence. The epithelial nature of the cells was proven by using an antikeratin antiserum. A high level of expression (close to 100% positive cells) of HLA class I antigens was observed on TEC at the beginning of the culture and remained unchanged for up to 12 days. In contrast, HLA class II antigen expression (85% DR+ and 75% DC+ cells on day 2) decreased gradually and reached very low levels (less than 5% DR+ or DC+) by day 7 of culture. This loss of class II antigen expression was not seen when cultures were performed in the presence of supernatants from activated T cells containing interferon-gamma (IFN-gamma). Furthermore, the presence of recombinant IFN-gamma (rIFN-gamma) in the medium from the onset of culture maintained HLA-DR and DC antigen expression on a high number of cells (comparable to that observed on day 2 of culture). A large percentage of rIFN-gamma-treated cells also showed intracytoplasmic HLA-DR antigen expression. Addition of rIFN-gamma at various times after the onset of the culture led to a reinduction of DR and DC antigen expression. This effect of rIFN-gamma was observed in 48 hr with concentrations as low as 10 IU/ml and was apparently specific for this IFN species, in that rIFN-alpha was unable to modify HLA class II antigen expression at concentrations up to 1000 IU/ml. The increased expression of HLA class II antigen was truly due to induction in individual TEC, rather than selection of class II-positive cells, because induction under the influence of IFN-gamma was reversible and occurred in the absence of proliferation in mitomycin-treated or gamma-irradiated cultures. Our results indicate that synthesis and membrane expression of class II HLA antigens are enhanced by IFN-gamma in TEC cultures. This finding raises the possibility that IFN-gamma participates in the mechanisms that assure the permanent expression of DR and DC antigens observed in TEC in vivo, with potentially important functional consequences in terms of education for self recognition.     

Partial IL-10 inhibition of the cell-mediated immune response in chronic beryllium disease.

Chronic beryllium disease (CBD) provides a human disorder in which to study the delayed type IV hypersensitivity response to persistent Ag that leads to noncaseating pulmonary granuloma formation. We hypothesized that, in CBD, failure of IL-10 to modulate the beryllium-specific, cell-mediated immune response would result in persistent, maximal cytokine production and T lymphocyte proliferation, thus contributing to the development of granulomatous lung disease. To test this hypothesis, we used bronchoalveolar lavage cells from control and CBD subjects to evaluate the beryllium salt-specific production of endogenous IL-10 and the effects of exogenous human rIL-10 (rhIL-10) on HLA expression, on the production of IL-2, IFN-gamma, and TNF-alpha, and on T lymphocyte proliferation. Our data demonstrate that beryllium-stimulated bronchoalveolar lavage cells produce IL-10, and the neutralization of endogenous IL-10 does not increase significantly cytokine production, HLA expression, or T lymphocyte proliferation. Second, the addition of excess exogenous rhIL-10 partially inhibited the beryllium-stimulated production of IL-2, IFN-gamma, and TNF-alpha; however, we measured no change in T lymphocyte proliferation or in the percentage of alveolar macrophages expressing HLA-DP. Interestingly, beryllium salts interfered with an IL-10-stimulated decrease in the percentage of alveolar macrophages expressing HLA-DR. We conclude that, in the CBD-derived, beryllium-stimulated cell-mediated immune response, low levels of endogenous IL-10 have no appreciable effect; exogenous rhIL-10 has a limited effect on cytokine production and no effect on T lymphocyte proliferation or HLA expression.     

Interferon-gamma regulates the T cell response to precursor nevi and biologically early melanoma

To examine the potential regulatory role of interferon-gamma in the cellular immune response to melanoma and its precursor lesions, we have tested the capacity of this lymphokine to enhance HLA class II antigen-dependent T lymphocyte blastogenesis, its in vitro production by autologous T cells stimulated by melanoma, and its presence in melanocytic lesions in situ. Cell lines derived from a dysplastic nevus, a radial growth phase primary tumor, a vertical growth phase primary, and metastatic lesions were induced by recombinant interferon-gamma to express increased amounts of HLA class II antigens. Such cells were then examined in radioimmunoassay for expression of HLA-DR antigens and in co-culture for their ability to stimulate proliferation of autologous T cells. Interferon-gamma treatment of melanocytic cells increased their expression of HLA-DR antigens threefold to sixfold. In parallel with these findings, co-culture of T cells with interferon-treated cells of a dysplastic nevus and a radial phase melanoma led to augmented T cell incorporation of tritiated thymidine, and this stimulation was inhibited with a monoclonal antibody to HLA-DR antigens. Despite augmented expression of HLA class II antigens (HLA-DR, -DQ, and -DP), vertical growth phase and metastatic melanoma cells failed to stimulate autologous T cells. When T cells were co-cultured with stimulating melanoma cells, culture supernatants contained significantly increased amounts of interferon-gamma (12 U/ml) in comparison with supernatants of T cells alone (4 U/ml). No interferon was detectable in cultures of melanoma cells alone. To link these in vitro phenomena to in situ events, we used murine monoclonal antibodies to interferon-gamma, the interleukin 2 receptor, and HLA-DR antigens in an immunoperoxidase system to detect interferon production and lymphocyte activation in frozen sections of lesions representative of melanocytic tumor progression. In these studies, precursor dysplastic nevi and radial phase melanomas contained the highest numbers of activated lymphocytes and stained positively for interferon-gamma. These results suggest that interferon-gamma plays a central role in the regulation of the cellular immune response to melanoma. It is produced by T cells, likely activated by tumor antigens seen in the context of HLA class II antigens. In turn, interferon-gamma production enhances expression of HLA class II antigens by melanoma and precursor cells, and such enhancement is associated with additional T cell activation in a positive feed-back loop.     

Generation of melanoma-specific, cytotoxic CD4(+) T helper 2 cells: requirement of both HLA-DR15 and Fas antigens on melanomas for their lysis by Th2 cells

Recognition of melanoma antigens by HLA class-II-restricted CD4(+) T lymphocytes has been investigated. Two cytotoxic CD4(+) T cell lines were established by stimulating PBLs from a melanoma patient with either parental or IFN-gamma-transduced autologous tumor cells. These T cells secreted IL-4, but not IL-2, IFN-gamma, or TNF-beta, in response to the autologous melanoma cells, suggesting that they belong to the Th2 subtype. Their cytotoxicity was directed against the IFN-gamma-transduced melanoma cells and was HLA-DR-restricted. The autologous and two allogeneic IFN-gamma-modified melanoma cell lines shared melanoma antigen(s) presented in the context of HLA-DR15. HLA-DR15(+) nonmelanoma cells were resistant targets indicating that the shared antigen(s) is melanoma associated. Parental autologous and HLA-DR-matched allogeneic melanoma cell lines, displaying low levels of HLA-DR antigens, induced Th2 proliferation and cytokine release, but were insensitive to lysis prior to upregulation of HLA-DR and Fas antigens by IFN-gamma. Cytolysis was inhibited by anti-HLA-DR and by anti-Fas antibodies, suggesting that the cytolysis is mediated via the Fas pathway. While small amounts of HLA-DR15 molecules on melanoma cells are sufficient for Th2 proliferation and cytokine release, higher amounts of HLA-DR15 and the expression of Fas are required for CD4(+)-mediated lysis.     

Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.     

Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum

Objectives: Translational research using adult stem cells derived from various tissues has been highlighted in cell‐based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta‐derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. Materials and methods: Placental extract, extracted using water‐soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up‐take, PAS (Periodic Acid‐Schiff) staining and urea production were also analysed. Results: The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte‐specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. Conclusions: Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human‐based medium, in translational research for regenerative medicine.     

Deficient IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-gamma to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-gamma production by allogenic adult CD4(+) T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-gamma production by T cells.     

Functional abolition of monocyte HLA-DR by aldehyde treatment. A novel approach to studies of class II restriction elements in antigen presentation

The effect of low concentration aldehyde treatment (0.0012 to 0.005%) on the expression of HLA-DR Ag by human monocytes was investigated. This treatment was shown to selectively abolish the expression of HLA-DR determinants defined by a monomorphic mAb (YE2.36) in a rosette assay. The expression of class I MHC Ag and Fc gamma R remained unaffected. As a result, the presentation of the recall Ag tetanus toxoid and streptokinase-streptodornase (SK-SD) to freshly isolated autologous T cells and T cell clones was completely inhibited. Increasing the concentration of aldehyde to 0.05% consistently produced partial restoration of Ag-presenting capacity. Low dose aldehyde treatment did not affect monocyte viability or membrane turnover. Thus, aldehyde-treated monocytes produced a second generation of HLA-DR and expression was almost completely restored to normal after 24 h of culture. The presentation of monocyte class II Ag as alloantigens was also inhibited by low dose aldehyde treatment but inhibition was much less marked when monocytes were aldehyde treated at 2 h rather than at 24 h of culture. This is consistent with the reexpression of HLA-DR which occurred readily in the first 24 h of culture and much less readily thereafter. Low dose aldehyde treatment did not affect Ag uptake and processing. However, monocytes which had been pulsed with Ag, aldehyde-treated to abolish HLA-DR, and then cultured to allow regeneration of HLA-DR could present Ag only when given a second Ag pulse, suggesting that once the association between microbial Ag and HLA-DR had formed the Ag was not then free to reassociate with novel HLA-DR. Low dose aldehyde treatment did not affect monocyte IL-1 production, neither did it inhibit the detection of HLA-DR by soluble mAb in FACS analysis. These results are consistent with the view that low dose aldehyde treatment disrupts the tertiary structure of human Ia molecules such that allostimulatory determinants and restriction elements for exogenous Ag are rendered inaccessible to T lymphocyte receptors and to cell-bound anti-DR mAb in the rosette assay, although DR determinants may remain accessible to soluble mAb.     

Modulation of human lymphocyte marker expression by gamma irradiation and mitomycin C.

gamma irradiation (GR) or mitomycin C (MC) treatment of stimulator cells is frequently used to achieve unidirectionality of response in the mixed lymphocyte reaction. As GR differs from MC in the pathways used to block lymphocyte replication, this study analyzes the effects of these modalities upon the expression of various differentiation and Class II major histocompatibility antigens on lymphocytes cultured for 24, 48, and 72 hr. There was a decrease in the mean density of HLA-DR expression on CD3+ and on CD8+ cells at 24, 48, and 72 hr after exposure to GR (42 and 35, 60 and 69, and 26 and 49%, respectively) or to MC (26 and 11, 26 and 18, and 46 and 30%, respectively). There was a parallel decrease in the levels of the corresponding cell subsets when compared with control cultured cells not exposed to GR or MC. In contrast, the density of HLA-DR markers on CD3-negative cells was increased at 24, 48, and 72 hr of culture following exposure to GR (73, 82, and 102%, respectively) or to MC (9, 45, and 80%, respectively). There was a more profound decrease in CD3+, CD8+, and CD19+ cell subset levels and in the density of the corresponding markers in GR-treated cells than in those of cells exposed to MC when the results were compared with those of untreated cultured control cells. Although GR appears to exert a more profound effect than MC, the results indicate that both modalities have the capacity to reduce the density of polymorphic determinants on Class II (HLA-D region)-encoded molecules on T (CD3+ and CD8+) and B (CD19+) cells which are known to trigger potent MLR responses. Both modalities may therefore affect profoundly the relative strength of MLC responses and the derived measurements of the degree of HLA Class II compatibility between stimulator and responder cells.     

Production of autoantibodies against citrullinated antigens/peptides by human B cells

Autoantibodies against citrullinated protein Ags (ACPA) are associated with the development of rheumatoid arthritis (RA). This immune response against citrullinated protein Ags, which is thought to be facilitated by certain MHC HLA-DR alleles, is highly specific for this disease and has been speculated to be involved in the pathogenesis. We have previously studied cultures of B cells for the production of Abs against HLA Ags. The aim of the current study was to examine the role of B cells in the production of ACPA in patients with RA. Peripheral blood B cells from RA patients and healthy people were cultured with EL4-B5, a murine cell line expressing human CD40L, and with T cell factors to stimulate the in vitro production of Abs by B cells isolated from peripheral blood. ACPA were produced by cultured B cells from RA patients, as determined by reactivity to cyclic citrullinated peptide (CCP). The results showed that 22% of the healthy persons tested also had B cells that could produce ACPA. Patients with HLA-DR alleles carrying the RA-associated shared epitope appeared to have more B cells with autoimmune potential for CCP than those without such HLA alleles (odds ratio 8.1, p = 0.001). In healthy individuals, anti-CCP-producing B cells were also observed more frequently if the RA-associated MHC genes were present (odds ratio 8.0, p = 0.01). Analysis of B cells in cultures may shed light on the interaction of genetic and environmental factors in the development of RA.     

Differential induction by immune interferon of the gene products of the HLA-D region on the melanoma cell line MeWo and its metastatic variant MeM 50-10

This study has shown for the first time an association between the metastatic properties of two autologous melanoma cell lines and their susceptibility to induction of HLA class II Ag by IFN-gamma. After in vitro incubation with IFN-gamma the melanoma cell line MeWo did not acquire reactivity with anti-HLA class II antibodies, whereas its metastatic variant MeM 50-10 did. The differential susceptibility to induction of HLA class II Ag on the two cell lines cannot be accounted for by either differences in the number and affinity of IFN-gamma receptors or in the sensitivity to IFN-gamma, but most likely reflects an intrinsic property of each cell line. Serologic and immunochemical investigations with anti HLA-DR, DQ, and DP mAb have indicated that only HLA-DR Ag are induced by IFN-gamma on MeM 50-10 cells. Northern blot analysis with HLA-DR beta, DQ beta, and DP beta probes suggest that different mechanisms underlie the differential susceptibility to induction by IFN-gamma of the gene products of the HLA-D region. The regulatory mechanism(s) that control the expression of HLA class II Ag appear to be different from those controlling the expression of the melanoma-associated Ag tested, inasmuch as the modulation of the latter by IFN-gamma did not differ on the two melanoma cell lines.     

Upregulation of HLA class II, but not intercellular adhesion molecule 1 (ICAM-1) by granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3) in synergy with dexamethasone.

In this study we have compared the effects of granulocyte macrophage colony stimulating factor (GM-CSF) on purified normal blood monocytes, with two other haemopoietic growth factors, Interleukin (IL-) 3 and Macrophage (M-)CSF on HLA class I, class II and intercellular adhesion molecule 1 (ICAM-1) expression in the presence and absence of dexamethasone (Dex). IL-3 alone, like GM-CSF, was a weak inducer of HLA class II expression but in combination with Dex markedly enhanced HLA-DR, DP and DQ expression. Similar changes were observed for HLA class I expression. The response to both IL-3 and GM-CSF was not additive in the presence of an optimal concentration of one cytokine and titrating concentrations of the other indicating that they may use common receptors and signal transduction mechanisms. Although IL-3 or GM-CSF alone also enhanced ICAM-1 expression, Dex inhibited both constitutive and the cytokine induced expression of this antigen. In contrast M-CSF, in the presence or absence of Dex, failed to enhance ICAM-1, HLA class I or II expression. These observations further highlight differences between the effects of the haemopoietic growth factors GM-CSF and IL-3 versus M-CSF in the regulation of monocyte function. Finally, the distinct effect of a combination of glucocorticoids with GM-CSF or IL-3 to induce high levels of HLA expression on human monocytes suggests they may have an important role during inflammatory conditions in vivo.     

Granulocyte macrophage colony stimulating factor (GM-CSF) biological actions on human dermal fibroblasts.

Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GM-CSFR) on human normal skin fibroblasts from healthy subjects (NFPC) and on a human normal fibroblast cell line (NHDF) and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GM-CSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM) components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC) is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF) cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such "differentiation" is an important event induced by such cytokine.     

Monocyte Chemoattractant Protein-1 Secreted by Decidual Stromal Cells Inhibits NK Cells Cytotoxicity by Up-Regulating Expression of SOCS3

Background

Decidual stromal cells (DSCs) are of particular importance due to their pleiotropic functions during pregnancy. Although previous research has demonstrated that DSCs participated in the regulation of immune cells during pregnancy, the crosstalk between DSCs and NK cells has not been fully elucidated. To address this issue, we investigated the effect of DSCs on perforin expression in CD56⁺ NK cells and explored the underlying mechanism.

Methodology/Principal Findings

Flow cytometry analysis showed perforin production in NK cells was attenuated by DSC media, and it was further suppressed by media from DSCs pretreated with lipopolysaccharide (LPS). However, the expression of granzyme A and apoptosis of NK cells were not influenced by DSC media. ELISA assays to detect cytokine production indicated that monocyte chemoattractant protein-1 (MCP-1) in the supernatant of DSCs conditioned culture significantly increased after LPS stimulation. The inhibitory effect of DSC media on perforin was abolished by the administration of anti-MCP-1 neutralizing antibody. Notably, reduced perforin expression attenuated the cytotoxic potential of CD56⁺NK cells to K562 cells. Moreover, Suppressor of cytokine signaling 3 (SOCS3) expression in NK cells was enhanced by treatment with MCP-1, as measured by RT-PCR and western blot. Interestingly, MCP-1-induced perforin expression was partly abolished by the siRNA induced SOCS3 knockdown. Western blot analysis suggested that both NF-κB and ERK/MAPKs pathway were involved in the LPS-induced upregulation of MCP-1 in DSCs.

Conclusions/Significance

Our results demonstrate that LPS induces upregulation of MCP-1 in DSCs, which may play a critical role in inhibiting the cytotoxicity of NK cells partly by promoting SOCS3 expression. These findings suggest that the crosstalk between DSCs and NK cells may be crucial to maintain pregnancy homeostasis.     

Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells

In the present study, we show that human self-MHC-reactive (autoreactive) T cell clones are functionally distinct from Ag-specific T cell clones. Self-MHC-reactive T cells exhibited helper function for B cell Ig production when cultured with non-T cells alone, and they exhibit suppressor function when cultured with PWM- or rCD40 ligand (rCD40L)-activated non-T cells, whereas tetanus toxoid (TT)-specific clones exhibited only helper function in the presence of TT with or without PWM or rCD40L. Addition of neutralizing Abs to the cultures showed that the suppression was mediated by TGF-beta but not by IL-10 or IFN-gamma. The self-MHC-reactive clones also inhibited proliferation of primary CD4+ T cells and TT-specific T cell clones, but in this case the inhibition was mediated by both IL-10 and TGF-beta. In further studies, the interactions between self-MHC-reactive T cell clones and non-T cells that led to suppressor cytokine production have been explored. We found that prestimulation of non-T cells for 8 h with PWM or for 48 h for rCD40L results in non-T cells capable of inducing self-MHC-reactive T cell to produce high levels of TGF-beta and IL-10. In addition, these prestimulation times coincided with peak induction of HLA-DR and costimulatory B7 molecule (especially CD86) expression on B cells. Finally, addition of CTLA-4/Fc or blocking F(ab')2 anti-CTLA-4 mAb, plus optimally stimulated non-T cells, to cultures of self-MHC-reactive clones inhibited the induction of TGF-beta but not IL-10 or IFN-gamma production. In summary, these studies show that activated self-MHC-reactive T cells have the cytokine phenotype of Th3 or T regulatory cell 1 and thus may be important regulatory cells that mediate oral and peripheral tolerance and prevent the development of autoimmunity.     

Cyclosporine A Enhances Th2 Bias at the Maternal-Fetal Interface in Early Human Pregnancy with Aid of the Interaction between Maternal and Fetal Cells

Our previous study has demonstrated that cyclosporine A (CsA) administration in vivo induces Th2 bias at the maternal-fetal interface, leading to improved murine pregnancy outcomes. Here, we investigated how CsA treatment in vitro induced Th2 bias at the human maternal-fetal interface in early pregnancy. The cell co-culture in vitro in different combination of component cells at the maternal-fetal interface was established to investigate the regulation of CsA on cytokine production from the interaction of these cells. It was found that interferon (IFN)-γ was produced only by decidual immune cells (DICs), and not by trophoblasts or decidual stromal cells (DSCs); all these cells secreted interleukin (IL)-4, IL-10, and tumor necrosis factor (TNF)-α. Treatment with CsA completely blocked IFN-γ production in DICs and inhibited TNF-α production in all examined cells. CsA increased IL-10 and IL-4 production in trophoblasts co-cultured with DSCs and DICs although CsA treatment did not affect IL-10 or IL-4 production in any of the cells when cultured alone. These results suggest that CsA promotes Th2 bias at the maternal-fetal interface by increasing Th2-type cytokine production in trophoblasts with the aid of DSCs and DICs, while inhibiting Th1-type cytokine production in DICs and TNF-α production in all investigated cells. Our study might be useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.     

Human myoblasts as antigen-presenting cells.

Human myoblasts, cultured from muscle and purified to greater than 95%, were investigated for their capacity to act as facultative APC. The myoblasts reacted with antidesmin mAb and had the capacity to fuse into multinucleated myotubes in appropriate medium. The expression of HLA class I, HLA-DR, HLA-DP, HLA-DQ, intercellular adhesion molecule-1 (ICAM-1/CD54), lymphocyte function-associated (LFA) molecules LFA-1 (CD11a/CD18), LFA-2 (CD2), and LFA-3 (CD58) was investigated by FACS analysis before and after induction for various times with human rIFN-gamma, TNF-alpha, or both. Without cytokine induction, myoblasts expressed only HLA-class I and LFA-3. IFN-gamma alone or in combination with TNF-alpha induced the expression of HLA-DR and ICAM-1 reaching a plateau after 48 h, followed by HLA-DP and even later HLA-DQ. TNF-alpha alone induced only ICAM-1. The functional capacity of myoblasts to present Ag to CD4+ T cells was investigated using autologous T cell lines specific for tuberculin, tetanus toxoid, and human myelin basic protein. Noninduced myoblasts or myoblasts treated with TNF-alpha alone could not present any of these Ag to the T cells. However, myoblasts treated with IFN-gamma induced Ag-specific proliferation. In the presence of relevant Ag, myoblasts were killed by the T cells as observed by microscopy and measured by 51Cr release. Ag-specific T cell proliferation and myoblast killing was inhibited in the presence of anti-DR mAb. These results suggest that human myoblasts may act as facultative APC during local immune reactions in muscle.     

Counter-antigen presentation: fibroblasts produce cytokines by signalling through HLA class II molecules without inducing T-cell proliferation

Fibroblasts are known to express histocompatibility leukocyte antigen DR (HLA-DR) molecules on their cell surface upon stimulation with interferon gamma (IFN- gamma), while the exact roles of HLA-DR on fibroblasts remain undetermined. To understand the role of HLA-DR molecules on fibroblasts, we examined whether: (1) fibroblasts act as antigen presenting cells (APC) which activate helper T (Th) cells; and/or (2) fibroblasts are activated via HLA-II molecules by making a T-cell receptor (TCR)-peptide-major histocompatibility complex (MHC) complex. We used Th(0) clone HT8.3, which recognizes an antigenic peptide (Ag53 p141-161) in the context of DRB1*1501, as well as IFN - gamma - treated and irradiated periodontal ligament fibroblasts (PDL) expressing DRB1*1501 molecules. When peptide-pulsed fibroblasts were co-incubated with HT8.3 treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. The antigen presenting capacity of these fibroblasts was evaluated by examining the proliferative responses of Th cells. Possible activation of fibroblasts by stimulation via HLA-DR molecules was evaluated by quantitating secreted cytokines in the supernatants after 18-h culture with or without anti-HLA-DR monoclonal antibody (mAb) or emetine-treated HT8.3. Indeed, Th cells did not show proliferative responses when peptide-pulsed PDL were used as APC, whereas PDL produced larger amounts of interleukin (IL) 6, IL-8, monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation, normal T expressed and secreted (RANTES) compared with controls, when cultured with anti-HLA-DR mAb or emetine-treated HT8.3. These findings suggest that HLA-DR expressed on fibroblasts do not present antigens to induce T-cell proliferation, but may act as receptor molecules that transmit signals into fibroblasts, based on DR-peptide-TCR interaction, resulting in the secretion of several cytokine species.