Early Pregnancy Factor Enhances the Generation and Function of CD4+CD25+ Regulatory T Cells.

The mechanisms of fetal semi-allograft acceptance by the mother's immune system have been the target of many immunological studies. Early pregnancy factor (EPF) is a molecule present in the serum of pregnant mammals soon after conception that has been reported to have immunomodulatory effects. In the present study, we aimed to determine whether immune cells such as CD4+CD25+ regulatory T cells (Tregs) are involved in the suppressive mechanism of EPF. Accordingly, CD4+CD25- T cells were isolated from spleens of female C57BL/6 mice and stimulated with anti-CD3 antibody, anti-CD28 antibody and IL-2 in the presence or absence of EPF. Flow cytometry was used to analyze the differentiation of CD4+CD25- T cells to CD4+CD25+ Tregs. We thus found a remarkable rise in the Treg ratio in the EPF-treated cells. Higher mRNA and protein levels of fork head box P3 (Foxp3), a marker of the Treg lineage, were also observed in cells treated with EPF. Furthermore, the effect of EPF on Treg immunosuppressive capacity was evaluated. EPF treatment induced the expression of interleukin-10 and transforming growth factor ß1 in Tregs. The suppressive capacity of Tregs was further measured by their capability to inhibit T cell receptor-mediated proliferation of CD4+CD25- T cells. We thus found that EPF exposure can enhance the immunosuppressive functions of Tregs. Overall, our data suggest that EPF induces the differentiation of Tregs and increases their immunosuppressive activities, which might be an important mechanism to inhibit immune responses during pregnancy.

Soluble Immune Response Suppressor (SIRS): Reassessing the immunosuppressant potential of an elusive peptide.

A previously studied immunosuppressive cytokine, Soluble Immune Response Suppressor (SIRS), may have relevance to current studies of immune suppression in a variety of human disease states. Despite extensive efforts using experimental models, mainly in mice, much remains to be discovered as to how autoimmune cells in mice and humans escape normal regulation and, conversely, how tumor cells evade evoking an immune response. It is the contention of this commentary that the literature pre-2000 contain results that might inform current studies. The broadly immunosuppressive protein, SIRS, was studied extensively from the 1970s to 1990s and culminated in the determination of the n-terminal 21mer sequence of this 15kDa protein which had high homology to the short neurotoxins from sea snakes, that are canonical members of the three finger neurotoxin superfamily (3FTx). It was not until 2007 that the prophylactic administration of the synthetic N-terminal peptide of the SIRS 21mer, identical to the published sequence, was reported to inhibit or delay the development of two autoimmune diseases in mice: experimental allergic encephalomyelitis (EAE) and type I diabetes (T1D). These findings were consistent with other studies of the 3FTx superfamily as important probes in the study of mammalian pharmacology. It is the perspective of this commentary that SIRS, SIRS peptide and the anti-peptide mAb, represent useful, pharmacologically-active probes for the study of the immune response as well as in the potential treatment of autoimmune, inflammatory diseases and cancer.

The significance of heat-shock protein gp96 and its receptors' CD91 and Toll-like receptor 4 expression at the maternal foetal interface.

PROBLEM: Differences in the expression of gp96 and its receptors were analysed in normal and pathological human pregnancy. MATERIAL AND METHODS: Immunohistology and immunofluorescence of sections from decidual part of term placenta, first trimester normal decidua, missed abortion and blighted ovum decidua were performed together with reverse transcriptase-quantitative polymerase chain reaction and flow cytometry. RESULTS: In missed abortion, gp96 was intensively stained, when compared to normal early pregnancy. The intensity of CD91 and TLR4 was higher in the first trimester pregnancy and blighted ovum, when compared to missed abortion. Decidual part of the term placenta is invaded with gp96⁺ , CD91⁺ and TLR4+ trophoblast. Progesterone-induced blocking factor (PIBF) decreased the frequency of TLR4⁺ T lymphocytes, CD91⁺ T, natural killer (NK) and mature dendritic cells after an 18-h culture. Decidual mononuclear cells (DMCs) treated with PIBF down-regulated CD91, TLR4 and gp96 gene expression. CONCLUSION: The presence of gp96, CD91 and TLR4 at the maternal-foetal interface provides a molecular basis for their interaction, particularly in the absence of PIBF.

DC maturation and function are not altered by melanoma-derived immunosuppressive soluble factors.

BACKGROUND: Although melanoma can elicit robust tumor antigen-specific immune responses, advanced melanoma is associated with immune tolerance. We have previously described several mechanisms of melanoma-induced immunosuppression, including the skewing of the immune response towards a Th2 cytokine profile and the induction of regulatory T cells. Since dendritic cells (DCs) are potentially important players that can direct other cells of the immune system towards a cytotoxic, humoral, or regulatory phenotype, we hypothesized that melanoma-produced factors directly affect the maturation and function of DCs, influencing the nature and magnitude of the resulting immune response. MATERIALS AND METHODS: To test this hypothesis, immature myeloid-derived DCs (mdDCs) were derived with cytokines from CD14+ peripheral blood mononuclear cells (PBMCs) and exposed to 20% melanoma-conditioned media (MCM). After 2 d, the expression of maturation markers and the function of these mdDCs, measured by cytokine production, the amount of endocytosis, expression of the inhibitory molecule indoleamine 2,3-dioxygenase (IDO), and the ability to stimulate T cells were determined. RESULTS: We found that incubation with MCM did not inhibit the expression of maturation markers or IDO, the production of cytokines, the amount of antigen uptake, or the ability to induce T cell proliferation in mixed-lymphocyte reactions by mdDC. CONCLUSIONS: These results suggest that the immunosuppressive effects of melanoma-produced factors are independent of directly measurable changes in mdDC function or maturation in vitro.

Immunoregulatory properties of heme oxygenase-1.

Heme oxygenase-1 (HO-1) is one of the three isoforms of the heme oxygenase enzyme that catabolyzes the degradation of heme into biliverdin with the production of free iron and CO. HO-1 is induced by its substrate and by other stimuli, including agents involved in oxidative stress and proinflammatory cytokines as well as several anti-inflammatory stimuli. A growing body of evidence points toward the capacity of this molecule to inhibit immune reactions and the pivotal role of HO-1 in inflammatory diseases. We will first review the physiological role of HO-1 as determined by the analysis of HO-1-deficient individuals. This will be followed by an examination of the effect of HO-1 within immunopathological contexts such as immune disorders (autoimmunity and allergy) or infections. A section will be devoted to the use of an HO-1 inducer as an immunosuppressive molecule in transplantation. Finally, we will review the molecular basis of HO-1 actions on different immune cells.

Immunosuppressive mechanisms during viral infectious diseases.

For a virus to establish persistence in the host, it has to exploit the host immune system such that the active T-cell responses against the virus are curbed. On the other hand, the goal of the immune system is to clear the virus, following which the immune responses need to be downregulated, by a process known as immunoregulation. There are multiple known immunoregulatory mechanisms that appear to play a role in persistent viral infections. In the recent past, IL-10 and PD-1 have been identified to be playing a significant role in the regulation of antiviral immune responses. The evidence that viruses can escape immunologic attack by taking advantage of the host's immune system is found in LCMV infection of mice and in humans persistently infected with HIV and HCV. The recent observation that the functionally inactive T-cells during chronic viral infections can be made to regain their cytokine secretion and cytolytic abilities is very encouraging. Thus, it would be likely that neutralization negative immune regulation during persistent viral infection would result in the preservation of effector T-cell responses against the virus, thereby resulting in the elimination of the persistent infection.

Recombinant EPF/chaperonin 10 promotes the survival of O4-positive pro-oligodendrocytes prepared from neonatal rat brain.

Chaperonin 10 (cpn 10) is a small heat-shock protein that is usually intracellular. Early pregnancy factor (EPF), a biologically active protein that was first described in the serum of pregnant mammals, is homologous to cpn 10. EPF/cpn 10 has been reported to have effects on immunomodulation and cell survival and to inhibit activation of toll-like receptors by lipopolysaccharide. We found that recombinant EPF/cpn 10 was able to suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, which is a disease causing inflammation and demyelination of the brain and spinal cord. This beneficial effect could be due to anti-inflammatory and/or cell survival properties of EPF/cpn 10. We aimed to assess the effects of cpn 10 on cells of the oligodendrocyte lineage because oligodendrocytes are the brain cells that produce myelin and that are depleted in multiple sclerosis. Two forms of recombinant EPF/cpn 10 were prepared in the pGEX expression system and in the baculovirus expression system. Purified O4(+) pro-oligodendrocytes were prepared from the brains of day-old Wistar rats and isolated by cell sorting with flow cytometry. Single cells were dispensed into micro-well plates and tested for survival in the presence of a range of concentrations of the two forms of cpn 10. We also studied the effects of bFGF, PDGF, IGF-1 and insulin as controls. With cpn 10 present, there was enhanced survival of O4(+) cells.

Ingested (oral) SIRS peptide 1-21 suppresses type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) is a chronic disorder that results from autoimmune destruction of the insulin-producing pancreatic beta cell. The nonobese diabetic (NOD) mouse is a model of the human autoimmune disease T1D. Soluble immune response suppressor (SIRS) is a nonspecific protein suppressor of immune response produced by immunomodulatory T cells stimulated by type I interferon (IFN). SIRS inhibits antibody responses in vivo, lipopolysaccharide (LPS)-induced fever, and delayed-type hypersensitivity (DTH) responses. Previous investigators have isolated the N-terminal sequence of SIRS protein consisting of 21 amino acids. Mice ingesting 1 microg SIRS peptide 1-21 showed significant delayed onset of T1D and a decreased frequency of T1D compared with mock-fed and 10-microg-fed mice and a significant decrease in islet inflammation. There were significant decreases in islet lymphocyte chemokine production of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein-1 gamma (MIP-1 gamma), regulated upon activation, normal T cell-expressed, and presumably secreted (RANTES), and stromal cell-derived factor-1 (SDF-1) in the SIRS-fed mice, factors important in migration of inflammatory cell into the islets. Ingested (oral) SIRS peptide inhibits clinical T1D by decreasing target organ cellular migration of islet destructive populations by suppression of islet lymphocyte chemokine secretion.

How do mesenchymal stromal cells suppress T cells?

Accumulating information indicates that mesenchymal stem or stromal cells (MSCs) are immunomodulatory, but the data to explain the observations are frequently conflicting. In this issue of Cell Stem Cell, Ren et al. (2008) provide evidence for a possible underlying mechanism of MSC-mediated T cell suppression. A perspective for considering these interesting observations is discussed.

Immunoneutralization of endometrial monoclonal nonspecific suppressor factor beta (MNSFbeta) inhibits mouse embryo implantation in vivo.

Successful embryo implantation and pregnancy in mammals depends on the establishment of immune tolerance between the maternal immune system and fetal cells. Monoclonal nonspecific suppressor factor beta (MNSFbeta), a cytokine produced by suppressor T cells in various tissues, possesses an antigen-nonspecific immune-suppressive function, and may be involved in the regulation of the uterine immune response during embryo implantation. In this study, anti-MNSFbeta IgG administered directly into the uterine lumen, significantly inhibited mouse embryo implantation in a dose-dependent manner in vivo, and this effect was reversed by co-administration of recombinant MNSFbeta. The effects of anti-MNSFbeta IgG on the gene pattern profiles in mouse uterine tissues were examined by cDNA microarray and several changes were confirmed by real-time PCR. Anti-MNSFbeta IgG caused up-regulation (> or = 2-fold) of 71 known genes and 17 unknown genes, and decreased expression (> or = 2-fold) of 74 known genes and 43 unknown genes, including several genes previously associated with embryo implantation or fetal development. Most of the known genes are involved in immune regulation, cell cycle/proliferation, cell differentiation/apoptosis, and lipid/glucose metabolism. These results demonstrate that MNSFbeta plays critical roles during the early pregnancy via multiple pathways.

[The influence of TLSFJM on the proportion of Th1/Th2-like cell subsets].

AIM: To explore the influence of TLSF(JM) on the proportion of alloantigen activated Th1 and Th2-like cell subsets. METHODS: TLSF(JM) or IL-4 was added to mixed lymocyte reaction(MLR) system. The influence of TLSF(JM) on the proportion of Th1 and Th2-like cell subsets was analyzed by intracellular immunofluorescence staining and FACS. RESULTS: In the TLSF(JM) group, the proportion of IFN-gamma(+) cells differentiated from activated lymphoblast descended from 49.8% to 43.1%, IL-4(+) cells from 75.4% to 43.7% and IL-6(+) cells from 67.8% to 52.6%. The similar tendency was also observed in the unactivated small lymphocytes. CONCLUSION: TLSF(JM) can inhibit both the Th1 and Th2-like cell subsets, but mainly inhibit the Th2-like cell subset, thereby reducing the proportion of Th2-like cell subsets.

Characterization of ubiquitin-like polypeptide acceptor protein, a novel pro-apoptotic member of the Bcl2 family.

Monoclonal nonspecific suppressor factor (MNSF) is a cytokine with antigen nonspecific suppressive activity. MNSFbeta (a subunit of MNSF) is a 14.5 kDa fusion protein consisting of a protein with 36% identity with ubiquitin and ribosomal protein S30. The ubiquitin-like segment (Ubi-L) may be cleaved from MNSFbeta in the cytosol. Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T-helper type 2 clone, D.10 cells. In this study, we purified a 33.5 kDa Ubi-L adduct from D.10 cell lysates by sequential chromatography on DEAE, anti-(Ubi-L) Ig-conjugated Sepharose, and hydroxylapatite. MALDI-TOF-MS fingerprinting revealed that this Ubi-L adduct consists of an 8.5 kDa Ubi-L and a Bcl2-like protein, murine orthologue of a previously cloned human BCL-G gene product with pro-apoptotic function. Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen. In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and interferon gamma-activated D.10 cells. The 33.5 kDa Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue. The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells. These results suggest that the post-translational modification of Bcl-G by Ubi-L might be implicated in T-cell activation.

A protein in pig spleen similar to immune suppressive protein of stress.

AIM: To purify a protein in pig spleens, which was similar to immune suppressive protein of stress (ISPS), and characterize its properties and functions. METHODS: 1) Pig spleen was extracted in dilute hydrochloric acid. 2) The extract was ultra-filtrated for having high molecular weight proteins (Mr>30 000). 3) The filtrates were purified with FPLC affinity chromatography. 4) The elute from FPLC was used for T-lymphocyte proliferation and ELISA test. 5) Lastly, SDS-PAGE was used to determine the molecular weight and purity of the final product. RESULTS: A protein purified from pig spleen (the pig ISPS homologue) inhibited concanavalin A (Con A)-induced mouse lymphocyte proliferation. The molecular weight of this protein was about Mr 190 000. It has a stronger selectivity against T-lymphocyte line such as Jurkat cell line and mastocyte line (P8l5) and has a weaker inhibitory activity on macrophage line (U937). CONCLUSION: A protein similar to rat/mouse ISPS was found in pig spleen. This may provide an opportunity to study its roles in tumors and autoimmune diseases.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E. coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding approximately 42 and 36mg EPF from 300ml bacterial and 1L Sf9 cultures, respectively. The preparations were highly purified (#10878;99% purity on SDS-PAGE for the bacterial products and #10878;97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immunosuppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity.

Allo-immunization elicits CCR5 antibodies, SDF-1 chemokines, and CD8-suppressor factors that inhibit transmission of R5 and X4 HIV-1 in women.

Studies in humans suggest that allo-immunization induces CC-chemokines, CD8-suppressor factors (SF) and anti-HIV immunity. Here we report that allo-immunization with unmatched leucocytes from partners of women with recurrent spontaneous abortion elicits specific antibodies to the CCR5 receptor. Such antibodies inhibit replication of M-tropic HIV-1 (R5) and MIP-1beta-mediated chemotaxis. These CCR5 antibodies were also found in the sera of multiparous women that were naturally immunized by semi-allogeneic fetal antigens. The specificity of these antibodies was demonstrated by adsorption with CCR5 transfected HEK-293 cells, a baculovirus CCR5 preparation and a peptide of the 2nd extra-cellular loop of CCR5. Allo-immunization also stimulated increased concentrations of the CXC chemokine, SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 (X4) replication. We suggest that allo- immunization may elicit (a) CC chemokines, CCR5 antibodies and CD8-SF that inhibit M-tropic HIV-1 infection and (b) the CXC chemokine SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 infection.

[A preliminary study on the immune suppressive protein of stress in human tonsil].

Our previous work demonstrated that under the conditions of restraint stress and under the control of central nervous system (CNS), an immune suppressive protein of stress (ISPS) was generated in peripheral lymph tissue and released into the blood stream, acting as an immune suppressor. In the present work, a protein similar to ISPS was found in human tonsil (a peripheral lymph tissue). Human tonsil was homogenized and the extract was prepared. It was found that lymphocyte proliferation was significantly suppressed by the extract. The suppression induced by the extract was partially reversed by the monoclonal antibody against ISPS (2C4). In ELISA test, the extract was able to bind to the monoclonal antibody. By immunohistochemistry, many ISPS positive cells were found in human tonsil. The ISPS positive cells were also found in human lymph nodes. Taken together, all the results demonstrate that a protein similar to ISPS may exist in human peripheral lymphoid tissue.

Transforming growth factor-beta suppresses interferon-gamma-induced toxoplasmastatic activity in murine macrophages by inhibition of tumour necrosis factor-alpha production.

Activation of macrophages plays an important role in the host resistance against intracellular pathogens. Various mechanisms are employed to control the activation processes and limit tissue damage by factors produced by activated macrophages. One of these mechanisms is the production of macrophage-deactivating cytokines, such as tumour growth factor (TGF)-beta. The present study concerns the effects of TGF-beta on interferon (IFN)-gamma-induced activation of murine macrophages with respect to induction of toxoplasmastatic activity, and production of tumour necrosis factor (TNF)-alpha, prostaglandin E2 (PGE2) and reactive nitrogen intermediates (RNI). IFN-gamma activation of macrophages resulted in inhibition of T. gondii proliferation [mean fold increase (FI) = 1.8, control mean FI = 7.0]; polymyxin B had no effect on this activation. The IFN-gamma-induced toxoplasmastatic activity of macrophages was inhibited by TGF-beta (mean FI = 6.3), which was also found for the IFN-gamma-induced production of TNF-alpha, RNI and PGE2 by macrophages. We found that PGE2, which has macrophage deactivating properties, was not involved in the inhibition of macrophage activation by TGF-beta. The deactivating activities of TGF-beta on the IFN-gamma-induced toxoplasmastatic activity and production of RNI are mediated by inhibition of production of TNF-alpha. Addition of exogenous TNF-alpha during the incubation of macrophages with IFN-gamma and TGF-beta abrogated the deactivating activity of TGF-beta. In sum, the results demonstrate that inhibition of TNF-alpha production is a key factor in the TGF-beta-induced suppression of macrophage activation with respect to toxoplasmastatic activity and RNI production.

Regeneration and tolerance factor of the human placenta induces IL-10 production.

Regeneration and tolerance factor (RTF) was originally identified in the placenta of mice and the isolated protein shown to have suppressive effects. In these studies, the gene cloned from thymus tissue was mapped to human chromosome 12. The role of recombinant RTF on cytokines was examined. In addition, we examined the human placenta by immunohistochemistry for RTF expression. RTF was expressed at the peripheral layer of cytotrophoblast in 7-9-week-old placentas. Using the RTF gene sequence, a recombinant protein was prepared and shown to induce IL-10 production. These data indicate that RTF is expressed by the tissues most intimately involved at the maternal-fetal interface, and its biological activity is capable of producing the necessary immune response for initiating and maintaining the maternal-fetal relationship.

Protein tyrosine phosphorylation induced by ubiquitin-like polypeptide in murine T helper clone type 2.

Ubi-L, an isoform of the monoclonal nonspecific suppressor factor (MNSF), is an 8.5-kDa ubiquitin-like polypeptide. Ubi-L shows an antigen-nonspecific immunosuppressive action on various target cells including murine T helper type 2 clone, D10 cells. Most recently, we have characterized the biochemical nature of the receptor for Ubi-L. In this study, we observed that Ubi-L receptor ligation rapidly and transiently stimulated tyrosine phosphorylation of 65- and 31-kDa proteins in concanavalin A-activated D10 cells. The addition of neutralizing antibody to Ubi-L receptor inhibited the protein tyrosine phosphorylations and the Ubi-L-mediated suppression of IL-4 production by D10 cells. Genistein, a tyrosine kinase inhibitor, also reduced the induction of these protein tyrosine phosphorylations. IFNgamma, which is also known to inhibit the proliferative response of D10 cells, showed a synergistic effect with Ubi-L. Interestingly, IFNgamma enhanced the Ubi-L-induced tyrosine phosphorylation of the 31-kDa protein. These results suggest that tyrosine phosphorylation may be a key step in the initiation of the Ubi-L receptor-mediated transmembrane signaling.