Cloning of rhesus monkey LILRs.

Leukocyte Ig-like receptors (LILRs) are a family of receptors that have inhibitory and activating functions and widely expressed by lymphoid and myeloid cells. Here we report the identification of the rhesus monkey LILRs by screening of rhesus spleen and decidua cDNA libraries and RT-PCR cloning. We obtained eight different full-length clones with structural and functional diversity similar to human LILRs, including LILRs with immunoreceptor tyrosine-based inhibitory motifs, LILRs with truncated cytoplasmic tails containing positively charged arginine residues in the transmembrane domain, and putative soluble receptors lacking transmembrane or cytoplasmic domains. Characterization of rhesus LILRs will facilitate use of this non-human primate model for the study of the functional role(s) of LILRs, including immune regulation through interaction with non-classical MHC class I molecules.

Selective distribution and pregnancy-specific expression of DC-SIGN at the maternal-fetal interface in the rhesus macaque: DC-SIGN is a putative marker of the recognition of pregnancy.

We performed immunohistochemical analysis of DC-SIGN expression at the maternal-fetal interface at different stages of pregnancy in the rhesus monkey. Natural killer cells, monocytes and macrophages were observed in the nonpregnant endometrium, particularly in the luteal phase, and were increased in pregnant endometrium. No DC-SIGN+ cells were observed in the nonpregnant uterus. We observed decidual DC-SIGN+ cells within 1 week of implantation, and they increased in number during the first 5 weeks of gestation. DC-SIGN+ cells showed a clear differential distribution in the decidua in the first 2 weeks of pregnancy, being found only adjacent to the implantation site, in marked contrast to the widespread distribution of CD68+ macrophages and CD56+ NK cells throughout the endometrium. DC-SIGN+ cells also showed a more dendritic morphology than the general CD68+ cell population, and analysis of serial sections indicated an overlapping but not identical localization of these markers. Mature dendritic cells could not be detected as judged by total absence of immunostaining for CD83, CD86, DEC-205, or CD1a. DC-SIGN+ cells were defined as MHC class II+ and CD14+ by flow cytometry. We conclude that DC-SIGN expression is an early response by the primate maternal immune system to the implanting embryo. The selectively distributed population of DC-SIGN+ decidual leukocytes may represent a morphologically and phenotypically distinct subpopulation of decidual macrophages of early pregnancy that could contribute to the establishment of maternal-fetal immune tolerance.

Dynamic changes in primate endometrial leukocyte populations: differential distribution of macrophages and natural killer cells at the rhesus monkey implantation site and in early pregnancy.

The distribution of uterine leukocytes during the periimplantation period cannot be readily evaluated in human pregnancy. Using immunohistochemistry we examined the distribution of macrophages, natural killer (NK) cells, and T cells in the non-pregnant endometrium and in the decidua at early stages of implantation and pregnancy in the rhesus monkey. CD68+ macrophages, CD56+ lymphocytes and CD3+ T cells were present in the proliferative and secretory endometrium. The number of macrophages and CD56+ lymphocytes dramatically increased at implantation (day 14-15 of pregnancy) and continued to be high in early pregnancy decidua. Macrophages were conspicuously more numerous in proximity to implantation site (decidua basalis) as compared to sites peripheral to the developing placenta (decidua parietalis), and were found in close association with cytotrophoblasts adjacent to the decidua, as well as around arteries invaded by extravillous cytotrophoblasts. In contrast to macrophages, CD56+ lymphocytes were more evenly distributed throughout the decidua. Few CD3+ T cells were seen in pregnancy, being scattered in the endometrial stroma with occasional aggregate formation. The distribution of uterine leukocytes vis-à-vis trophoblasts at the rhesus monkey implantation site and in early pregnancy suggests different roles for macrophages and uterine NK cells in the response to trophoblast invasion.

Initiation of alcoholic fatty liver and hepatic inflammation with a specific recall immune response in alcohol-consuming C57Bl/6 mice.

Whether immunological responses are involved in initiation and progression of alcoholic liver disease is unclear. We describe a mouse model of alcoholic liver injury characterized by steatosis and hepatic inflammation initiated by a recall immune response. Mice immune to Listeria monocytogenes fed a liquid diet containing ethanol and challenged with viable bacteria developed steatosis within 24 h and, at a later time, elevated serum alanine aminotransferase levels, indicating more liver damage in this group. Listeria antigen also induced steatosis and increased serum alanine aminotransferase levels in immune ethanol-consuming mice. The production of tumour necrosis factor by a recall immune response in this model is a major, but not the only, component in initiation of alcoholic liver disease.

Phenotypic and functional characterization of rhesus monkey decidual lymphocytes: rhesus decidual large granular lymphocytes express CD56 and have cytolytic activity.

In this study, we carried out a phenotypic and functional characterization of lymphocytes isolated from the uterine endometrium of the pregnant rhesus monkey. A majority (80%) of these cells were CD56(bright+), CD3- had typical large granular lymphocyte/uterine natural killer (NK) cell morphology and contained numerous cytoplasmic granules. Flow cytometric evaluation showed that rhesus decidual CD56(bright+) cells shared other phenotypic features of human uterine NK cells, including low levels of CD45RA and CD62L expression. A majority of the rhesus uterine CD56(bright+) cells expressed low levels of CD 16 but were CD2-. In contrast, most rhesus CD16+ peripheral blood cells were CD56-. In addition to the primary population of CD56(bright+) cells, a minor subset of smaller and less granular CD56(intermediate+) decidual lymphocytes was identified, the majority of which were CD16-, CD2(+). Decidual CD56+ cells did not express monocyte/macrophage markers, including CD14, CD64 and CD68. Decidual lymphocytes effectively lysed K562, Raji and particularly 721.221 targets in cytotoxicity assays. Together, these results suggest that as in human pregnancy, rhesus decidual CD56(bright+) cells represent a distinct lymphocyte subset that belongs to the NK cell lineage.

Placental expression of the nonclassical MHC class I molecule Mamu-AG at implantation in the rhesus monkey.

During human implantation trophoblasts mediate attachment of the embryo to the uterine epithelium and invade and reorganize vessels of the maternal endometrium to initiate blood flow to the intervillous space. Expression of the nonclassical MHC class I molecule HLA-G by invading trophoblasts may play a central role in their protection from recognition by the maternal immune system; however, the ontogeny of trophoblast HLA-G expression during the earliest stages of implantation is difficult to evaluate in human pregnancy. We previously identified a novel nonclassical MHC class I molecule, Mamu-AG, which is expressed in the rhesus monkey placenta and shares many unique characteristics of HLA-G. Immunocytochemical analysis with a Mamu-AG-specific mAb and locus-specific in situ hybridization of rhesus implantation sites 7-12 days after embryo attachment (days 14-19 of pregnancy) demonstrated that Mamu-AG molecules are expressed predominantly in cytotrophoblasts invading the maternal vessels and endometrium, whereas syncytiotrophoblasts covering trophoblastic lacunae or newly formed chorionic villi remained largely Mamu-AG-negative. By day 36 of pregnancy, Mamu-AG glycoprotein also was expressed in villous syncytiotrophoblasts, and accumulation of Mamu-AG glycoprotein was noted at the border between maternal decidua and fetal trophoblasts. The ontogeny of a nonclassical MHC class I molecule at the implantation site supports the hypothesis that its expression is important for the establishment of maternal-fetal immune tolerance.

Analysis of blood lymphocyte subsets in children living around Chernobyl exposed long-term to low doses of cesium-137 and various doses of iodine-131.

Epidemiological studies have found that children living around Chernobyl have rates of respiratory tract illness that are higher than those seen in the area before the Chernobyl accident. The present study investigates the possible effects of radiation exposure on the composition of peripheral blood lymphocyte subsets in children living around Chernobyl. Two hundred nineteen healthy children and children suffering from recurrent respiratory diseases aged 6-14 years who received both low doses of radiation to the whole body from (137)Cs and various doses of radiation to the thyroid from (131)I as fallout from the accident were assessed 5 (1991) and 8-10 years (1994-1996) after the accident. A total of 148 healthy children and children suffering from recurrent respiratory diseases living in noncontaminated areas were also evaluated as controls. Children with recurrent respiratory diseases who lived around Chernobyl had a significantly lower percentage of T cells and a higher percentage of NK cells compared to control children with recurrent respiratory diseases during the study period. In contrast to the findings in 1991, a significant decrease in the percentage of helper-inducer cells was observed in children with recurrent respiratory diseases in 1994-1996. In contrast to 1991, there is a positive correlation between the percentage of helper-inducer cells, the helper-inducer/cytotoxic-suppressor cell ratio, and the dose of radiation to the thyroid of healthy children from (131)I in 1994-1996. There was a positive correlation between the dose of radiation to the thyroid from (131)I and the percentage of helper-inducer cells in children with recurrent respiratory diseases 5 years (1991) after the accident. Further, the dose of radiation to the thyroid from (131)I correlated negatively with the percentage of T and B cells and positively with the percentage of NK cells in children with recurrent respiratory diseases 8-10 years (1994-1996) after the accident. These results raise the possibility that long-term exposure to low doses of (137)Cs may have altered the composition of the T-cell subsets and NK cells in children with recurrent respiratory diseases. The differences in the composition of the peripheral blood lymphocyte subsets between healthy children and those with recurrent respiratory diseases may be attributed to long-term low-dose exposure of the whole body to radiation from (137)Cs and exposure of the thyroid to radiation from (131)I subsequent to the Chernobyl accident.

Mamu-I: a novel primate MHC class I B-related locus with unusually low variability.

The rhesus macaque is an important animal model for several human diseases and organ transplantation. Therefore, definition of the MHC of this species is crucial to the development of these models. Unfortunately, unlike humans, lymphocytes from a single rhesus macaque express up to 12 different MHC class I cDNAs. From which locus these various alleles are derived is unclear. In our attempts to define the MHC class I loci of the rhesus macaque, we have identified an unusual MHC class I locus, Mamu-I. We isolated 26 I locus alleles from three different macaque species but not from three other Cercopithecine genera, suggesting that the I locus is the result of a recent duplication of the B locus occurring after the divergence of macaques from the ancestor of the other extant Cercopithecine genera. Mamu-I mRNA transcripts were detected in all tissues examined and Mamu-I protein was produced in rhesus B lymphoblastoid cell lines. Furthermore, Mamu-I protein was detected by flow cytometry on the surface of human 721.221 cells transfected with Mamu-I. In contrast to the polymorphism present at this locus, there is unusually low sequence variability, with the mean number of nucleotide differences between alleles being only 3.6 nt. Therefore, Mamu-I is less variable than any other polymorphic MHC class I locus described to date. Additionally, no evidence for positive selection on the peptide binding region was observed. Together, these results suggest that Mamu-I is an MHC class I locus in primates that has features of both classical and nonclassical loci.

Tissue distribution of the mRNA for a rhesus monkey major histocompatibility class Ib molecule, Mamu-AG.

We identified recently a novel major histocompatibility complex (MHC) class I locus in the rhesus monkey, Mamu-AG, which is expressed in the placenta and encodes molecules that share unique characteristics of human HL4-G. We established locus-specific reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assays to determine whether Mamu-AG is expressed in other rhesus monkey tissues. With an RT-PCR assay, Mamu-AG mRNA was detected in placenta, amniotic membranes, kidney, spleen, eye, brain, lung, spinal cord, liver and occasionally heart, but was undetectable in lymph nodes, salivary glands, peripheral blood lymphocytes (PBL), large and small intestine, skeletal muscle or skin. Examination of endocrine organs demonstrated the presence of Mamu-AG transcripts in pituitary, testes, ovary and adrenal glands but not in pancreas or thyroid. Quantitative analysis using a ribonuclease protection assay demonstrated that the highest level of Mamu-AG mRNA expression was consistently in the placenta and amniotic membranes, while expression was moderate in a few tissues (testis, adrenal) and low to undetectable in all other tissues. These results suggest that the Mamu-AG mRNA, like the mRNA for the human MHC class Ib gene HLA-G, is expressed at high levels in the placenta, but also has restricted low-level expression in other tissues.

The rhesus monkey analogue of human lymphocyte antigen-G is expressed primarily in villous syncytiotrophoblasts.

The human placenta expresses the nonclassical major histo-compatibility complex (MHC) class I molecule, human lymphocyte antigen (HLA)-G, which may contribute to the establishment of maternal-fetal immune tolerance. Although the HLA-G ortholog of the rhesus monkey, Mamu-G, is a pseudogene, another nonclassical MHC class I locus, Mamu-AG, is expressed in the rhesus monkey placenta. Mamu-AG encodes MHC class I A locus-related molecules that exhibit all the characteristics of human HLA-G, including limited polymorphism and a truncated cytoplasmic domain. We have examined MHC class I glycoprotein and Mamu-AG mRNA expression in the rhesus placenta and in cultured trophoblasts. Immunocytochemical analysis of rhesus placental tissues with the W6/32 monoclonal antibody demonstrated a high level of MHC class I expression in villous syncytiotrophoblasts, whereas villous cytotrophoblasts were largely MHC class I negative. Only low levels of MHC class I expression were seen in extravillous cytotrophoblasts of cell columns and the trophoblastic shell. In situ hybridization demonstrated that Mamu-AG mRNAs were expressed at a high level in first-trimester villous syncytiotrophoblasts. MHC class I and Mamu-AG expression was significantly up-regulated during in vitro culture and differentiation of freshly isolated villous cytotrophoblasts into syncytiotrophoblasts. Preferential Mamu-AG expression in syncytiotrophoblasts suggests that rhesus monkey MHC class I-bearing trophoblasts could potentially interact with maternal peripheral blood lymphocytes rather than with uterine decidual lymphocytes as has been proposed for human trophoblasts.

131I dose-dependent thyroid autoimmune disorders in children living around Chernobyl.

We assessed the major lymphocyte subsets in the peripheral blood, thyroid ultrasonography, levels of serum autoantibodies to thyroglobulin (AbTg), thyroid hormones, and thyroid-stimulating hormone (TSH) in 53 children without any chronic diseases living continuously around Chernobyl. The subjects ranged in age from 7 to 14 years and had different doses of 131I to their thyroid. Healthy children living on noncontaminated areas were assessed as controls. The majority of children with doses of 131I had normal levels of thyroid hormones. However, the percentages of positive sera for AbTg, TSH levels, ultrasonographic thyroid abnormalities, and abnormal echogenicity were significantly higher in children with doses of 131I than in controls. The dose of 131I to thyroid correlated positively with serum AbTg levels, percentage of CD3+CD4+ cells, and CD3+CD4+/CD3+CD8+ cell ratio and negatively with number of CD3+CD8+ and CD3-/CD16, CD56+ cells. Thus, our study demonstrates an association between dose of 131I and autoimmune thyroid disorders in this population of children.

Analysis of blood lymphocyte subsets in children living on territory that received high amounts of fallout from Chernobyl accident.

The major lymphocyte subsets in the peripheral blood were assessed in 120 children 6-13 years old living on areas that received high levels of radioactivity as fallout after the Chernobyl nuclear power plant accident. Seventy-one of the children were suffering from recurrent respiratory disease (RRDC) and 49 were not (non-RRDC). As controls, a total of 87 RRDC and non-RRDC living on noncontaminated areas were evaluated. We did not find significant differences in major lymphocyte subsets between the values in non-RRDC living on radionuclide-contaminated areas and noncontaminated areas. However, RRDC living on radionuclide-contaminated areas had a significantly lower percentage of CD3+ T and CD3+CD4+ T-helper/ inducer cells compared to control RRDC. Furthermore, the decrease in percentage of CD3+CD4+ cells was more profound in RRDC living in radiation-contaminated settlements with an average summary dose (ASD) Cs-137(134) and Sr-90 for the population > 1.0 mSv than in RRDC living in contaminated settlements with an ASD Cs-137(134) and Sr-90 < 1.0 mSv. These data indicated that long-time exposure to small doses of radiation could affect the immune system in children living around Chernobyl.

Differential expression of CD45RA and CD45RO molecules on human decidual and peripheral blood lymphocytes at early stage of pregnancy.

PROBLEM: The use of monoclonal antibodies for CD45RA and CD45RO antigens is important in defining maturational and functional stages on lymphocytes. METHOD: To characterize distribution of two isoforms of CD45 antigen CD45RA and CD45RO on CD3+, CD4+, CD8+ and CD56+ lymphocyte subsets from first trimester human decidua, two-color flow cytometry were used. RESULTS: In decidua, there were much higher levels of CD45RO+ and much lower levels of CD45RA+ cells among CD3+, CD4+, and CD8+ cells as compared with peripheral blood samples of the same pregnant women. Only approximately 40% of CD56+ cells in decidua expressed CD45RA. Unlike peripheral blood, approximately 30% of decidual natural killer (NK) cells weakly stained with anti-CD45RO antibodies. Double-negative CD45RA- CD45RO- NK cells were also present in decidua. CONCLUSIONS: The significantly raised percentage of intradecidual T cells expressing CD45RO suggest decidual accumulation of antigen-committed memory cells. The patterns of CD45 isoforms expression on decidual CD56+ cells are consistent with hypothesis that uterine CD56+ lymphocytes are terminally differentiated cells of NK lineage.

Different pathways of in vitro ethanol-induced apoptosis in thymocytes and splenic T and B lymphocytes.

To investigate the intracellular pathways leading to ETOH-induced apoptosis, thymocytes and splenic T and B cells were cultured 16 h with or without ETOH and different stimuli, and apoptotic cell death was determined. At concentrations of 0.4%-2% in culture, ETOH induced apoptosis in all three types of cells, but it had a more profound effect on thymocytes and B cells as compared with its effect on T cells. In thymocytes, ETOH-induced apoptosis was abrogated by chelation of extracellular calcium with EGTA, and inhibition of protein synthesis with CHX, or of PKC with H7 but not of PKA with HA 1004. ETOH potentiated the apoptosis of thymocytes induced with the calcium ionophore A23187 and suboptimal doses of PMA, but it had negligible effect on dAMP- and PGE2-induced apoptosis of thymocytes. In contrast to findings in thymocytes, the ETOH-induced apoptosis of T and B cells was almost completely abrogated by PMA, but not by H7 or CHX. In spleen cells, calcium chelation with EGTA triggered apoptosis. ETOH significantly inhibited EGTA-induced apoptosis of B cells but had little effect on EGTA-induced apoptosis of T cells. IL-4 reduced the ETOH-induced apoptosis of B and T cells, but it was not effective in the prevention of apoptosis of thymocytes. Inhibition of the calcium-dependent neutral protease calpain I did not rescue cells from apoptosis. Moreover, treatment with CI-I potentiated ETOH-induced apoptosis in T cells. These results suggest that both thymocytes and splenic T and B cells have relevant apoptotic pathways that can be induced by ETOH, but the mechanisms of ETOH-induced apoptosis differ in these cells.