C.S. Lee

Comparative T‐Cell Responses During Pregnancy in Large Animals and Humans

PROBLEM: Placentation in different large animal species shows a remarkable diversity in the level of trophoblast invasion into the maternal endometrial tissues. We wish to determine the influence of implantation on T‐cell responses during pregnancy. 
METHOD OF STUDY: Review of the literature and current data. 
RESULTS: αβ‐TCR+ T cells are only prominent during early pregnancy in species with relatively non‐invasive placentation (pig and horse) but are rapidly downregulated in species with more invasive placentae. γδ‐TCR+ T cells are prominent in species with moderate trophoblast invasion (ruminants) where they increase dramatically during mid and late pregnancy. γδ‐TCR+ T cells remain prominent during late gestation in species with highly invasive placentation (humans) and, in addition, a distinct γδ T‐cell population is present in first trimester decidua where it may play a regulatory role in controlling natural killer cell activity. The γδ‐TCR+ population present in both ruminants and humans shows large granular morphology and contains antimicrobial proteins, suggesting their function may be to protect the uterine environment from infection during pregnancy and parturition. 
CONCLUSION: The comparative analysis of T‐cell responses during pregnancy in different large animal species supports an increasing role for cells of the innate immune response (NK and γδ T cells) and a downregulation of the adaptive immune response with increasingly invasive placentation.

Uropathogenic virulence factors in isolates of Escherichia coli from clinical cases of canine pyometra and feces of healthy bitches.

Escherichia coli is commonly isolated in canine pyometra, but little is known of the virulence factors that may be involved in the precipitation of this disease. The aim of this study was to compare the prevalence of uropathogenic virulence factor (UVF) genes in E. coli isolates from canine pyometra and from feces of healthy bitches to evaluate their role in the pathogenesis of pyometra. E. coli from 23 cases of canine pyometra and from the feces of 24 healthy bitches were analyzed, by polymerase chain reaction, for UVF genes associated with canine and human urinary tract infections (UTIs). The prevalences of UVFs in E. coli from canine pyometra were similar to that in canine and human uropathogenic E. coli. The prevalence of pap was greater (P=0.036) for E. coli from pyometra (52%) than for fecal isolates (21%), and the papGIII allele was present in all pap-containing isolates. The prevalences of genes for alpha-haemolysin and cytotoxic necrotising factor 1 were not significantly higher (P=0.075) in E. coli from pyometra than from feces. The proportion of pyometra strains with >or=3 UVFs was higher (P=0.039) than that of fecal strains, suggesting that possession of >or=3 UVF genes enhances the pathogenicity of the strain. Our findings demonstrate that E. coli associated with canine pyometra are similar to uropathogenic strains, and that operons that encode P fimbriae, alpha-haemolysin and cytotoxic necrotising factor 1 probably enhance the virulence and pathogenicity of the strain in the canine genital tract.

Inflammatory response in the pig uterus induced by seminal plasma

The immunological and physiological influence of seminal plasma on the local uterine environment was investigated by immunohistochemical and flow cytometrical studies on uterine tissues and lymph nodes taken from gilts after mating with a vasectomised boar and from control, unmated gilts. These studies revealed that mating with a vasectomised boar induces an acute transient inflammatory response in the endometrium resulting in marked changes in the presence and distribution of leukocytes and extensive proliferation of the endometrial glands. At the same time there was an increase in CD8L and sIg+ cells and an up-regulation of MHC class II and IL-2 receptor expression in the uterine lymph nodes of mated pigs. This would suggest that seminal plasma deposited in the uterus can activate cells in the local draining lymph nodes. Together, these results demonstrate in utero that pronounced immunological and physiological changes are induced in vivo by seminal plasma.

Cellular responses during liver fluke infection in sheep and its evasion by the parasite

The cellular immune response in sheep to an acute and chronic primary and an acute secondary liver fluke infection were examined by immunohistology of liver tissue and flowcytometry of lymphocytes from the draining hepatic lymph nodes. Ten days after primary infection, portal tract areas surrounding migratory tunnels were infiltrated with CD4+ and CD8+ lymphocytes with fewer B cells and T19+ T cells. Micro abscesses were distributed sporadically in the liver parenchyma and young flukes could be easily observed in the liver tissue free from inflammatory cells. More intensive infiltration of the portal tract areas was observed during a secondary liver fluke infection characterized by a pronounced increase in eosinophils, B cells and CD4+ T cells. In addition, there was an increase in MHC class II+ fibroblastic‐like cells surrounding the migratory tracts. In contrast to the primary infection, no young flukes were observed in the same tissue areas during the secondary infection. Chronic primary infections were characterized by perilobular fibrosis and a predominance of CD8+ and γδ‐TCR+ T19‐ T cells distributed within fibrotic strands. Distinct B cell follicles were observed in the fibrotic strands and near major bile ducts and necrotic patches. Pronounced lymphocyte infiltration could occasionally be observed surrounding liver fluke eggs lodged in liver tissue. A progressive increase in lymph node weight, cell number and CD4/CD8 ratio was observed in the acute and chronic primary infections. The role of the infiltrating cell populations and possible mechanisms of immune evasion by the parasite are discussed.

Cellular immune responses in the pig uterus during pregnancy

In pigs, little is known about the role of the uterine immune system during pregnancy. Immunohistochemical studies were conducted using a panel of monoclonal antibodies to pig leukocytes on uterine tissues taken from gilts after fertile mating and at different stages of pregnancy. Acute inflammation in the endometrium in response to fertile mating which included marked changes in the tissue and immune cell components of the endometrium was observed. Throughout pregnancy the pig uterus contained a substantial population of leukocytes. MHC class II staining was prominent in the endometrium at all stages examined and included macrophages, dendritic and fibroblast-like cells, lymphocytes and the endothelial lining of many uterine blood vessels. The majority of lymphoid cells were CD2+, indicating the prevalence of T cells. In early pregnancy specific changes were seen in the tissue distribution of uterine immune cells. Following placentation distinct cellular changes in the local immune cell environment of the uterus were also observed despite the non-invasive nature of the pig placenta. There appears to be suppression and activation of various immune cell components in the uteri of pregnant pigs. This phenomenon is presumably in response to foetal or trophoblast antigens, suggesting that the local immune system is involved in the uterine response to pregnancy.

Quantitative and Qualitative Changes in the Intraepithelial Lymphocyte Population in the Uterus of Nonpregnant and Pregnant Sheep

PROBLEM: Previous studies have revealed the presence of a unique population of CD45R + granulated cells in the sheep uterine epithelium. In the present study, dramatic changes in this cell population and in the nongranulated lymphocytes in the uterine and endometrial glandular epithelium of non‐cycling, cycling, pregnant, and postparturient sheep are described. In noncycling and cycling sheep, the granules in the granulated intraepithelial cells were small. From days 55 to 134 of pregnancy, the granules in these cells were large, and there was a significant increase (P < 0.01) in the proportion of this cell population in the uterine epithelium but not in the endometrial glandular epithelium located in the deeper region of the stroma. The number of these cells declined dramatically (P < 0.01) from 2 to 15 days after parturition. Both the tissue distribution and the time of activation of these cells suggests they are different from the granulated lymphocytes described in placentae of mice and man.

Involution of the sheep mammary gland

Changes in the ovine mammary gland epithelium during initiated involution were studied by light and electron microscopy. Apoptosis of the duct and alveolar epithelial cells was first identified at 2 d after weaning, reached a peak at 4 d and then progressed gradually thereafter. Apoptotic cells were phagocytosed by intraepithelial macrophages and alveolar epithelial cells. Occasional apoptotic epithelial cells were observed in the alveolar and duct lumina. The highly vacuolated cells in the alveolar and duct lumina were confirmed to be macrophages as they were CD45+, MHC class II+. Changes in myoepithelial cells involved shrinkage and extension of cytoplasmic processes into the underlying stroma and no apoptosis was observed. Regression of the blood capillaries was also by apoptosis. The resulting apoptotic bodies were either taken up by adjacent endothelial cells or were shed into the capillary lumen to be phagocytosed later by mural endothelial cells or blood monocytes. The mammary glands were completely involuted by 30 d after weaning. It was concluded that the mammary gland involutes by apoptosis, a process which allows deletion of cells without the loss of the basic architecture and the integrity of the epithelial lining of the gland.

Studies on the distribution of immune cells in the uteri of prepubertal and cycling gilts

To establish the cellular basis for the local immune response in the porcine uterus, immunohistochemical studies using a panel of monoclonal antibodies to pig leukocytes were conducted on uterine tissues from prepubertal and cycling gilts. In prepubertal uteri, neutrophils were the most predominant cell type, while MHC class II+ cells and CD2+ T lymphocytes were also common. At the early-stage of the oestrous cycle, CD2+ T cells were numerous in the endometrium, particularly in the uterine epithelium and subepithelial regions. However, by the mid-stage of the cycle there was a significant and dramatic fall in CD2+ T cells and other lymphocytes expressing the CD4, CD8 and CD1 phenotypes, MHC class II+ cells were predominant throughout the endometrium. During late oestrus there was a dramatic infiltration of neutrophils into the subepithelial stroma. A distinct increase in the CD2+ intraepithelial T lymphocyte population was also observed at this stage of the cycle. It was concluded that in the healthy, non-pregnant pig uterus T lymphocytes, macrophages and neutrophils were the prominent leukocyte cell types and their migration and distribution in the uterus was strongly influenced by the oestrous cycle. These immune cells may play an important interactive role in the cyclic cellular changes in both the structure and function of the endometrium. Furthermore, the leukocyte phenotypes found in the porcine endometrium indicate that a local cellular immune response could be elicited.

Leucocyte phenotypes in involuting and fully involuted mammary glandular tissues and secretions of sheep

Mammary glandular tissues and mammary secretions were obtained from sheep at 2–60 d after weaning to study the leucocyte phenotypes associated with mammary involution. From 2–4 d after weaning, neutrophils were the predominant leucocytes in the alveolar and ductal lumina. Lymphocytes were present in the alveolar and ductal epithelium, interalveolar and periductal areas. Most of the lymphocytes in the alveolar and ductal epithelium (IEL) were CD8+, some were CD45R+ and few were CD4+. In the periductal clusters and in the interalveolar areas most of the lymphocytes were CD4+. There was a significant increase (P < 0.05) in the percentages of CD45R+ granulated IEL from 2 to 7 d after weaning, and this paralleled the increase in the percentages of apoptotic cells in the glandular epithelium. By 7–60 d after weaning, most cells within the alveolar and ductal lumina were macrophages followed by predominantly CD8+ lymphocytes. CD8+ lymphocytes were still predominant in the alveolar and ductal epithelium while CD4+ cells were predominant in the interalveolar areas. Very few γδ+ T cells were observed at all the stages examined. The cells in the mammary secretions correlated with those observed in the alveolar and ductal lumina. At the early stages of involution, the neutrophils and macrophages were heavily laden with lipid droplets, casein and cellular debris. The most interesting feature was the presence of cells either with extensive cytoplasmic processes (LCA+ MHC class II+) or cytoplasmic veils (LCA+ MHC class II+CD1+), probably dendritic cells. It is concluded that the cellular constituents of the mammary gland at the latter part of involution may afford the mammary gland more resistance to infection than the lactating gland and the gland at early stages of involution. The CD45R+ IEL may trigger apoptotic cell death in the mammary glandular epithelium during mammary involution.

Characterization of a sheep trophoblast-derived antigen first appearing at implantation

A monoclonal antibody designated SBU-3 was produced by the fusion of mouse NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with sheep trophoblast microvilli. Lee et al (1985) have reported the immunohistological staining of sheep trophoblast with SBU-3 showing that, as early as 21 days of gestation, the monoclonal antibody recognizes an antigen restricted to the binucleate cells of the trophoblast which are located only at sites of invasion of the underlying uterine tissue. Subsequently the antigen appears in the maternal syncytial layer. Immunoprecipitation of 125I-labelled microvilli by SBU-3, characterization of the antigen on immunoblots, and biochemical analysis all suggest that this monoclonal antibody specifically recognizes a carbohydrate epitope on a series of glycoproteins of molecular weights between 30 000 and 200 000. SBU-3 antigen is present in allantoic fluid but is not detectable in any fetal or adult tissue studied, including maternal and fetal sera. It is suggested that this antigen may have a role in the placentation process.