Robbert Benner

Chorionic gonadotropin induces dendritic cells to express a tolerogenic phenotype

The pregnancy hormone human chorionic gonadotropin (hCG) has been suggested to play an immunoregulatory role in addition to its endocrine function, thus contributing to the prevention of fetal rejection. We hypothesized that hCG is involved in the maternal‐fetal immune tolerance by the regulation of dendritic cell (DC) function. Therefore, we studied the effect of hCG on DC maturation. Upon hCG treatment in combination with LPS, mouse bone marrow‐derived DC (BMDC) increased the ratio of IL‐10:IL‐12p70, down‐regulated TNF‐α, and decreased antigen‐specific T cell proliferation. Addition of hCG together with LPS and IFN‐γ blocked MHC class II up‐regulation, increased IL‐10 production, and decreased the antigen‐specific T cell proliferation by DC. Splenic DC showed similar results. Upon hCG treatment, IDO mRNA expression and its metabolite kynurenine were increased by LPS‐ and IFN‐γ‐stimulated DC, suggesting its involvement in the decreased T cell proliferation. To study the effect of hCG on DC differentiation from precursors, BMDC were generated in the continuous presence of hCG. Under this condition, hCG decreased cytokine production and the induction of T cell proliferation. These data are suggestive for a contribution of hCG to the maternal‐fetal tolerance during pregnancy by modifying DC toward a tolerogenic phenotype.

Inhibition of diabetes in NOD mice by human pregnancy factor

Clinical symptoms of Th1 mediated autoimmune diseases regress in many patients during pregnancy. A prominent feature of pregnancy is the presence of human chorionic gonadotrophin hormone (hCG) in blood and urine. In this report we tested the effect of clinical grade hCG (c-hCG) on the development of diabetes, a Th1 mediated autoimmune disease, in nonobese diabetic (NOD) mice. We show that treatment of NOD mice with c-hCG before the onset of clinical symptoms lowered the increased blood glucose levels, reversed the established inflammatory infiltrate of pancreatic tissue, and profoundly inhibited the development of diabetes for prolonged time. c-hCG also induced profound inhibition of the functional activity (i.e. production of IFN-gamma) of Th1 cells. Transfer of spleen cells from c-hCG-treated NOD mice into immunocompromised NOD.SCID mice inhibited the development of diabetes in these otherwise nontreated mice. This shows that the treatment of the donor NOD mice induced persistent changes in the immune system. The antidiabetic activity of c-hCG was not caused by heterodimeric hCG or its subunits. Instead, this antidiabetic activity resided in a fraction of c-hCG preparation that contains a 400-2000 Dalton natural (immuno) modulatory pregnancy factor (NMPF).

Frequencies of background immunoglobulin-secreting cells in mice as a function of organ, age, and immune status

Abstract The influence of hereditary absence of thymus and spleen upon the numbers, organ, and class distribution of background immunoglobulin Ig-secreting cells was studied in mice by means of the protein-A plaque assay. In young adult BALB/c mice the spleen contained the largest number of Ig-secreting cells (about 0.5% ). The absolute number of Ig-secreting cells in the spleen was larger than the estimate for all lymph nodes together. Between 8 and 40 weeks of age, the number of Ig-secreting cells in spleen and lymph nodes increased by a factor of 3, maximally. In the same period, the number of Ig-secreting cells in the bone marrow, however, increased by a factor of 20, so that it became the major site of Ig synthesis. Hereditary absence of the spleen did hardly or not at all affect the number of Ig-secreting cells in the other lymphoid organs. However, the athymic state did affect the organ distribution. The most consistent finding was the decreased number of Ig-secreting cells in the Peyers patches. The class distribution of Ig-secreting cells was found to be independent of the presence of the spleen, but did depend on the presence of the thymus. Athymic mice had a higher percentage of IgM-secreting cells and a lower percentage of IgA-secreting cells. The percentage of IgG1- and IgG2-secreting cells did not differ clearly between normal and athymic mice. Percent-wise, most IgM-secreting cells occurred in the spleen, whereas most IgG1-, IgG2-, and IgA-secreting cells occurred in the bone marrow, lymph nodes, and Peyers patches. The specificity repertoire of the background Ig-secreting cells was tested by determining the frequencies of IgM-producing cells with specificity for a panel of six different antigens. These frequencies ranged from 1 in 85 for nitroiodophenyl(NIP)-conjugated sheep erythrocytes (SRBC) till 1 in 1500 for unconjugated SRBC and were found to be the same for the spleen of germ-free and specific pathogen-free (SPF) C3H mice, and for spleen, bone marrow, and thymus of SPF C3H mice.

Inhibition of septic shock in mice by an oligopeptide from the β-chain of human chorionic gonadotrophin hormone

Human chorionic gonadotrophin (hCG) is a heterodimeric placental glycoprotein hormone required in pregnancy. In human pregnancy urine and in commercial hCG preparations (c-hCG) it occurs in a variety of forms, including breakdown products. Several reports have suggested modulation of the immune system by intact hormone, but such effects of breakdown products have not been reported. In a related article (Hum Immunol 62:1315, 2001), it is reported that a 400-2000 Dalton (Da) fraction from c-hCG and from human pregnancy urine inhibits Th1-mediated diabetes in NOD mice. The active component(s) were called natural (immuno)modulatory pregnancy factor(s) (NMPF). This study reports that a single treatment with the same low molecular weight NMPF fraction up to 24-h after high dose lipopolysaccharide (LPS) injection inhibited septic shock in mice. This counteracting effect of NMPF paralleled the downregulation of the effects of LPS on the production of macrophage migration inhibitory factor (MIF) by spleen cells, on the plasma level of liver aminotransferase, and on the expression of several splenic lymphocyte and macrophage surface markers. Based on the primary structure of the beta-chain of hCG a synthetic hexapeptide Valine-Leucin-Proline-Alanine-Leucine-Proline (VLPALP) was designed, which demonstrated it to have the same protective effects as the 400-2000 Da NMPF fraction. These results indicate a new strategy for the treatment of septic shock and the potential of therapeutic use of this synthetic oligopeptide.

IL-1β and IFN-γ induce the regenerative epidermal phenotype of psoriasis in the transwell skin organ culture system. IFN-γ up-regulates the expression of keratin 17 and keratinocyte transglutaminase via endogenous IL-1 production

Skin biopsies from healthy human skin and non‐lesional skin from patients with psoriasis were cultured for 24h and stimulated with interleukin‐1β(IL‐1β) and interferon‐γ (IFN‐γ) in a skin organ culture model and the induction of the psoriasiform regenerative epidermal phenotype was analysed using immunostaining. In the presence of IL‐1β, the psoriasiform regenerative epidermal phenotype was clearly induced. This involved strong up‐regulation of the expression of keratin 16, keratin 17, and keratinocyte transglutaminase (TGk) in the suprabasal layers, strong up‐regulation and a shift of the expression of keratin 5 and integrin β1from the basal to suprabasal keratinocytes, and induction of the expression of ICAM‐1 and HLA‐DR on basal keratinocytes. The effects of IL‐1β in the organ cultures of normal skin could be completely neutralized by anti‐IL‐1 polyclonal antibodies. The effects of IFN‐γ in healthy and non‐lesional psoriatic skin were qualitatively similar to those of IL‐1β. The IFN‐γ‐induced epidermal expression of keratin 17 and TGk could be completely blocked by culturing the biopsies in the presence of IL‐1ra or anti‐IL‐1 antibodies, while the induction of HLA‐DR and ICAM‐1 was not inhibited. The induction of the psoriasiform regenerative epidermal phenotype by IFN‐γ is partially mediated via endogenous epidermal IL‐1. Copyright

Antibody formation in mouse bone marrow: IV. The influence of splenectomy on the bone marrow plaque-forming cell response to sheep red blood cells

Abstract Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during the secondary response, it becomes the major center of activity containing IgM-, IgG-, and IgAPFC. In the present paper the influence of splenectomy was studied on primary and secondary PFC activity in the bone marrow. Differences in primary and secondary bone marrow PFC responses are probably related to the presence of B and T memory cells in situ . Therefore the effect of splenectomy on the appearance of B and T memory cells in the bone marrow was also investigated. Splenectomy before intravenous (iv) immunization with 4 × 10 8 SRBC prevented any primary PFC activity in the bone marrow. The influence of splenectomy before priming on secondary PFC activity in the bone marrow depended on the priming dose of SRBC. Splenectomy before priming with 10 7 SRBC iv completely prevented IgM-, IgG-, and IgA-PFC activity in the bone marrow upon subsequent boosting with 4 × 10 8 SRBC iv. By means of cell transfer experiments it was shown that after splenectomy no B or T memory cells appeared in the bone marrow after priming with 10 7 SRBC iv. The effect of splenectomy on both secondary PFC activity and appearance of B and T memory cells in the bone marrow could be partly overcome by iv priming with a higher antigen dose of 4 × 10 8 SRBC. Cell transfer experiments showed that splenectomy before priming with 10 7 SRBC iv not only interfered with the appearance of B and T memory cells in the bone marrow, but also with the appearance of B memory cells in peripheral lymph nodes, mesenteric lymph node, Peyers patches, thymus, and blood. This indicates that after immunization with 10 7 SRBC iv all B and T memory cells which appear at extra splenic sites, are generated in the spleen. Immunization of splenectomized mice with 4 × 10 8 SRBC iv did induce the appearance of B memory cells in peripheral lymph nodes, mesenteric lymph node, Peyers patches, thymus, and blood. We suggest that small amounts of a high dose of iv inoculated SRBC can induce the generation of B memory cells in lymph nodes and Peyers patches.

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Abstract Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10 6 SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10 8 SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10 6 SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.

Chorionic gonadotropin can enhance innate immunity by stimulating macrophage function

Human chorionic gonadotropin (hCG) is a placental glycoprotein, mainly secreted by trophoblasts during pregnancy. Its function in endocrine regulation has been well documented, but its immunological role is still largely unclear. For a successful pregnancy, an effective innate immunity is needed to protect the mother and fetus against infection, while maintaining tolerance against the paternal antigens of the fetus. The aim of this study was to investigate the effect of hCG on the function of macrophages (Mϕ), which are major players in the innate response. hCG treatment of IFN‐γ‐primed Mϕ resulted in increased production of NO, reactive oxygen species, IL‐6 and IL‐12p40, and enhanced phagocytosis of apoptotic cells. hCG treatment did not affect the induction of allogeneic T cell proliferation by IFN‐γ‐primed Mϕ. The observed effects were receptor‐mediated and involved the protein kinase A signaling pathway, as indicated by blocking studies using specific inhibitors. In vivo thioglycollate‐elicited Mϕ also exhibited increased phagocytic ability upon IFN‐γ activation and hCG treatment. In conclusion, hCG enhances Mϕ functions involved in innate immunity, while the capacity to stimulate allogeneic T cells remains unchanged.

Improvement of the protein A plaque assay for immunoglobulin secreting cells by using immunoglobulin-depleted guinea pig serum as a source of complement.

This paper describes a modification of the protein A hemolytic plaque assay for the enumeration of immunoglobulin (Ig)-secreting cells independent of antibody specificity of the Ig. This assay was originally developed by Gronowicz et al. (1976), and is based upon binding of the Fc portion of IgG to protein A. Ig-secreting cells are mixed with protein A-coated sheep erythrocytes, developing rabbit anti-Ig antiserum and guinea pig serum as a source of complement. This mixture is either pipetted between two microscope slides, or added to agarose and plated on a petri dish or microscope slide. The hemolytic plaques are enumerated after incubation at 37 degrees C. Here we show that purification of the guinea pig complement over a Sepharose-protein A column in order to eliminate the IgG fraction facilitates plaque formation. This modification reduces the incubation period required for plaque formation, and yields a higher number of, and more discrete plaques, than the original method.

Suppression of antigraft immunity by preimmunization. I. Kinetic aspects and specificity.

Intravenous injection of 2,000 rad of irradiated allogeneic cells can suppress the development of antigraft delayed-type hypersensitivity (DTH) to major and minor histocompatibility (H) antigens which normally arises after s.c. immunization. Secondary type DTH responses to minor H antigens were also largely suppressed by an i.v. injection of irradiated allogeneic cells 1 week preceding the s.c. priming injection. The extent of suppression of primary DTH to allogeneic H-2-incompatible cells depended on the dose of i.v. injected irradiated cells. After a dose of 1 × 107 irradiated spleen cells i.v., the suppression persisted for at least 40 days. Intravenous injection of cells incompatible for minor H antigens could not suppress the DTH to H-2 alloantigens and vice versa. Suppression of DTH to H-2 alloantigens was haplotype specific. Proliferation studies indicated that the immunosuppressed mice do not respond upon s.c. immunization with an increased proliferative activity in the draining lymph nodes, in contrast to nonsuppressed mice. The data suggest that i.v. preimmunization with allogeneic cells induces specific suppression of antigraft immunity acting at the induction stage of the immune response.