Impact of tissue enzymatic digestion on analysis of immune cells in mouse reproductive mucosa with a focus on γδ T cells.

Mucosal tissues are enriched in γδ T lymphocytes, which maintain epithelial homeostasis, however, the homeostatic mechanisms are still incompletely understood. To elucidate their role in the tissue integrity governance within the female genital mucosa we employed flow cytometry, which is a powerful tool used for the characterization of tissue-resident immune cells, however, often requiring cell release upon tissue enzymatic disaggregation. Here, we analyzed the impact of various proteolytic enzymes in their ability to effectively isolate viable immune cells from the reproductive system of non-pregnant mice. Murine vaginas and uteri were digested using commercially available enzyme blends (liberases) and single enzymes (dispase II and collagenase IV). Among tested enzymes, liberases released the highest number of cells from digested tissues while dispase II and collagenase IV led to a significant decrease in the number of isolated live cells. Also, liberases had only minor detrimental effects on cell viability and detection of CD45, CD3ε, γδ TCR and CD11c positive cells. We found that a single liberase blend called Liberase TL was the most suited for the analysis of γδ T cells in the reproductive tract. By examining two distinct phases of the estrous cycle - estrus and diestrus, characterized by high and low epithelial stratification, respectively, we showed that higher numbers of γδ T lymphocytes were present in the latter cycle phase in vagina and uterus. Interestingly, the diestrus-associated increase in γδ T lymphocyte number was also observed in reproductive tract draining lumbar lymph nodes but not in more distant, inguinal lymph nodes. Our data indicate that enzymes used for reproductive mucosa digestion have profound effects on the cell viability and isolation efficiency, which consequently influence the phenotypic and quantitative analysis of immune cells.

The reproductive cycle is a pathogenic determinant during gonococcal pelvic inflammatory disease in mice.

Women with asymptomatic Neisseria gonorrhoeae infection are at risk of developing pelvic inflammatory disease (PID) if the bacteria ascend from the endocervix into the uterus and oviducts. Factors that affect disease severity, ranging from mild discomfort to severe inflammation, pain, and infertility, remain elusive. Herein we perform direct transcervical inoculation of N. gonorrhoeae into the uterus of mice to establish an infection that leads to PID. Profoundly different disease outcomes were apparent at different stages of the reproductive cycle. Mice that were infected during the diestrus stage of the reproductive cycle displayed extensive gonococcal penetration into the submucosa, severe inflammation, and clinical signs reflecting discomfort. Meanwhile, infection during the intervening estrus stage showed only modest effects. Furthermore, a gonococcal-specific humoral response was only elicited following the penetrative upper genital tract (UGT) infection during diestrus but not estrus. Strikingly, the potential for antibodies to contribute to protection during re-infection also depends upon the reproductive stage, as antigonococcal antibodies within the genital tract were markedly higher when mice were in diestrus. Combined, this work establishes a robust new model reflecting gonococcal PID in humans and reveals how the reproductive cycle determines the pathogenic outcome of gonococcal infections of the UGT.

Quantity of IL-2, IL-4, IL-10, INF-γ, TNF-α and KC-like cytokines in serum of bitches with pyometra in different stages of oestrous cycle and pregnancy.

The occurrence of the pyometra is most common in the first half of the dioestrus when there is decreased cellular immunity associated with increased serum concentration of progesterone in females. The aim of this study was to determine the immunological profile of bitches with pyometra, studying serum levels of IL-2, IL-4, IL-10, IFN-γ, KC-like and TNF-α and comparing them with those of healthy bitches in anoestrus, dioestrus and pregnant. Forty females were divided into four experimental groups: group 1 (G1): with pyometra (n = 10); group 2 (G2): bitches in the second week of gestation (n = 10); group 3 (G3): in anoestrus (n = 10); and group 4 (G4): in dioestrus (n = 10). The serum levels for IL-2, KC-like, INF-γ and TNF-α were similar for all experimental groups. The values obtained for IL-10 were found increased (p < 0.001) in animals in dioestrus and pyometra compared with females in anoestrus and pregnant, and the levels of IL-4 observed were significantly greater (p < 0.001) in bitches with pyometra when compared with others. The cytokine profile in animals with pyometra is similar to bitches in dioestrus for IL-10 and had increase in IL-4 for bitches with pyometra, which represents an anti-inflammatory these cases. This suggests the presence of an immunosuppressive state in both cases, which may explain the propensity of bitches in dioestrus to be affected by pyometra and the severity of the disease on these animals.

Characterisation of lymphocyte subsets in the equine oviduct.

REASONS FOR PERFORMING STUDY: The equine oviduct is the site of fertilisation and location of embryonic development during the first 5 or 6 days. It therefore has an important influence on mare fertility. Although histopathological changes have been described previously, there is limited information regarding lymphocyte subtypes present in the mucosa of the normal equine oviduct. OBJECTIVES: To characterise the distribution of CD3+, CD4+, CD8+ and B lymphocytes in the equine oviduct from inseminated mares during oestrus and dioestrus, and from noninseminated mares during the immediate post ovulatory period. METHODS: Oviductal tissues were collected from noninseminated mares at oestrus (> 30 mm follicle, n = 4), at Day 1 post ovulation (n = 3) and at dioestrus (Day 7 post ovulation; n = 4). Oviducts were also collected from inseminated mares at Days 1, 2, and 3 post ovulation (n = 4 for each period). Cross-sections of tissues from the ampullar-isthmic junction from each oviduct were snap frozen and cryostat sections stained by the immunoperoxidase technique with monoclonal antibodies directed against equine lymphocyte surface markers for B cells as well as CD3+, CD4+ and CD8+ cells. RESULTS: In all oviductal sections examined, B cells were rare whereas T cells were relatively abundant. The predominant cell type found was the CD8+ phenotype, with a lesser number of CD4+ cells. Among mares, individual variation was large; therefore, although breeding status and stage of oestrous cycle appeared to alter lymphocyte populations, these differences were not significant. CONCLUSIONS AND POTENTIAL RELEVANCE: A population of CD3+, CD4+ and CD8+ cells exists within the mucosal region of the equine oviduct. The density of these cells is similar to that described in the human oviduct. Their function is not currently known, but they may be involved with modulation of the maternal response to the presence of spermatozoa or the early conceptus within the equine oviduct. As our capacity to differentiate these cell types improves, along with the ability to identify the specific cytokines they produce, their functional significance will become more apparent.

Distribution of eosinophils and mast cells in the cervical tissue of non-gravid mares during dioestrus.

Little is known about the local cellular immune system of the equine cervix. In this study we characterize the distribution of eosinophilic granulocytes and mast cells in the cervical tissue of non-gravid mares during dioestrus. For this purpose cervices of 10 mares were histologically examined after Sirius red and Toluidine blue staining. Both cell types could be found in the lamina propria mucosae. In the tunica muscularis the two cell populations were only rarely detected. No cells could be found in the epithelium mucosae. There was a decreasing cell density from the vaginal to uterine side of the cervix (p < 0.01). Therefore, eosinophils might be a part of the local cellular immune system of the equine cervix during dioestrus, unlike in animals studied so far as cows and rats.

Cytokine profile of mouse vaginal and uterus lymphocytes at estrus and diestrus.

It is known that sex hormones regulate IgA and IgG levels in the female reproductive tract. Moreover, antigen presentation by uterine and vaginal epithelial cells is also under strict hormonal control. The effect of the estrous cycle on cytokine secretion by vaginal and uterine lymphoid cells has been examined in mice using simultaneous staining for cytoplasmic cytokines and surface markers after ex vivo culture with PMA/ionomycin in the presence of Brefeldin A, and flow cytometry analysis. Two different mice strains, BALB/c and C57BL/6 mice, were used. The most relevant finding was the increase in the proportion of vaginal cells secreting IFN-gamma at diestrus in both strains of mice. Other cytokines (IL-2 and IL-4) as well as some T cell subsets seemed to be modified in a strain dependent fashion. Data also suggest that NK cells are at least partially responsible for IFN-gamma secretion. Our data indicate that vaginal and uterus lymphoid cells isolated at diestrus were in vivo activated to secrete cytokines after ex vivo culture. IFN-gamma seems to be the key cytokine, since it increases in both strains of mice.

Effect of ovarian hormones on periodical changes in immune resistance associated with estrous cycle in the beagle bitch.

In bitches, the onset of pyometra, an infection of the uterus, characteristically occurs in the first half of the diestrous stage in the estrous cycle, in which the blood concentration of progesterone peaks and that of estradiol-17beta is lowest. To investigate the immunological mechanisms governing stage-specific onset of pyometra, peripheral blood mononuclear cells (PBMNCs) were collected from beagle bitches during different stages of the estrous cycle and examined using various immunological assays. When we examined the proliferative response of PBMNCs to PYO-252, that is a clone of Escherichia coli isolated from the uterus of a dog afflicted with pyometra, the response of PBMNCs significantly decreased in the first half (day 10) of diestrus, but increased in proestrus/estrus. No significant differences were observed in the responses to concanavaline A between stages of the cycle. Throughout the estrous cycle, canine PBMNCs did not respond to lipopolysaccharide derived from E. coli. The response of PBMNCs collected in anestrus to PYO-252 was significantly enhanced upon the addition of estradiol-17beta to the culture. In contrast, these responses were significantly suppressed in the presence of progesterone. Progesterone progenitor or metabolite molecules, which have a low affinity for the progesterone receptor, did not affect proliferative responses. Expression of gamma interferon (IFNgamma) in response to PYO-252 was also significantly enhanced by estradiol-17beta, but suppressed by progesterone. This evidence suggests that in the first half of the diestrous stage, suppressed activity of cellular immunity results from increasing progesterone concentration and minimal estrogen release. This marked decrease of immune resistance allows the expansion of E. coli, which enter the uterine cavity through the loosened cervical canal during estrus, leading to pyometra onset.

Macrophage clearance of 125I-labelled polyvinyl pyrrolidone in the horse: effect of ovarian steroids and persistent endometritis.

The rate of clearance of 125I-labelled polyvinyl pyrrolidone (PVP) from blood was measured in mares as an indicator of macrophage function. In three out of four cycling mares, PVP clearance was slower during oestrus than dioestrus. Similarly, administration of oestrogen to four ovariectomised mares tended to depress PVP clearance compared with clearance from the same mares before they received oestrogen. However, the effect of oestrogen was not statistically significant. Mares susceptible to persistent endometritis had rates of PVP clearance which were similar to those of genitally normal mares.

T and B lymphocyte populations in uterus draining lymph nodes at estrus and diestrus in Wistar rats.

In this paper, data are presented on the characterization of uterus draining lymph nodes (UDLN) B and T lymphocytes of virgin rats at estrus (E) and diestrus (D). We established that the T/B lymphocyte relationship in the UDLN is less than one at estrus and more than one at diestrus. This is due to a decrease in the percentage and in the total number of mature T cell population in association with a decrease in the percentage of the OX8 subset in the UDLN at estrus. This situation may be related to an increase in estrogens known to produce thymic involution. These changes were not observed when we studied peripheral (popliteal) lymph nodes. The changes observed in the UDLN T cell population at estrus could be under hormonal control and we think that this condition may be important to prepare the immune system for an eventual pregnancy.

A dioestrous increase in thymocyte proliferation during the oestrous cycle.

Coitus, which precedes internal fertilisation, is a unique physiological event which allows motile allogeneic spermatozoa to enter the female host and invade her tissues. The cyclic cellular proliferation observed in the thymus of the female rat may be an important preparation of her immune system for this event.

Opsonins of Streptococcus in uterine flushings of mares susceptible and resistant to endometritis: control of secretion and partial characterization.

The release of opsonins into the uterine lumen of mares susceptible or resistant to endometritis was examined after intrauterine inoculation of a filtrate of Streptococcus culture fluid or vehicle. Uterine flushings were collected at 0.5 hour before and 2, 4, 6, 8, and 24 hours after inoculation on day 2 or 3 of estrus and on day 7 or 8 after ovulation. Amounts of opsonins in flushings were quantified as the H2O2 produced by leukocytes incubated with flushings-opsonized bacteria, compared with H2O2 produced by leukocytes incubated with nonopsonized bacteria. Opsonin values in flushings increased (P less than 0.025) in all mares after inoculation of filtrate or vehicle. For mares resistant to endometritis, opsonin values were greater at diestrus than at estrus. The opposite was true for mares susceptible to endometritis, resulting in a status (susceptible vs resistant) X stage of cycle interaction (P less than 0.025). Overall, opsonins were higher (P less than 0.05) in flushings of mares susceptible to endometritis than in flushings of mares resistant to endometritis, but this difference was only apparent at estrus. Preliminary characterization of opsonins in uterine secretions by ammonium sulfate fractionation and gel filtration indicated that opsonins were mainly associated with an ammonium sulfate-soluble fraction of high molecular weight (greater than 4 X 10(6] and an ammonium sulfate-precipitable fraction that was associated with immunoglobulin G.