Search

Recognition of the mucosal portal of entry for many infectious diseases and of the relevance of mucosal immune response to protection has encouraged the development of vaccines administered by mucosal routes, principally oral and intranasal, for stimulation of intestinal and nasopharyngeal lymphoid tissues respectively. The oral route is problematic in cattle and other ruminants where antigen degradation in the rumen is likely, prior to transit to the intestine. On the other hand, rumination can be exploited for exposure of nasopharyngeal tissues during cudding if vaccine antigen is expressed by a fibrous feed like alfalfa. An increase in anti-leukotoxin (Lkt) IgA was demonstrated in nasal secretions of calves following feeding of alfalfa expressing a truncated Lkt50 from Mannheimia haemolytica, and there is evidence suggesting that such vaccination may protect against experimentally induced pneumonia. Intranasal vaccination is an alternative approach for use in pre-ruminating calves. Intranasal administration of ISCOMs carrying soluble antigens of M. haemolytica, including native Lkt, induced Lkt specific IgA in nasal secretions after vaccination at 4 and 6 weeks of age. Subcutaneous (s.c.) administration of the same vaccine induced Lkt specific IgG in both serum and nasal secretions, whereas s.c. administration of a commercial M. haemolytica vaccine did not. Regardless of the vaccination strategy employed it is difficult to assess the immunogenicity of mucosally administered vaccines because production of secreted antibodies tends to be transient, and they do not persist on the mucosal surface in the absence of ongoing antigenic stimulation. An additional challenge is demonstration of vaccine efficacy in response to experimental infection. Protection of the mucosally vaccinated animal will most probably result from recall response, which may not amplify sufficiently to counter the effects of experimental pulmonary delivery of a large bolus of virulent bacteria, even though the response would suffice over the more prolonged and gradual infection that occurs in natural induction of pneumonia.

Functions of uterine natural killer (uNK) cells during mouse pregnancy are maintenance of decidua basalis and promotion of uterine spiral artery modification, a process that results in thin-walled, dilated, elongated arteries with lowered resistance. Murine models indicate spiral artery modifications are triggered by release of the cytokine interferon (IFN)-gamma from uNK cells. The purpose of this study was to determine if human IFN-gamma could induce structural changes in the unmodified spiral arteries found in pregnant, alymphoid (uNK-, NK-, T-, B-) mice. Spiral arteries in pregnant Rag2 null/common cytokine chain (c) gamma null mice were modified, in a dose response manner, by daily injections of rhIFN-gamma. Administration of low dose LPS did not induce morphologically recognized structural changes. These findings are key in building humanized murine models of pregnancy since they suggest Rag2 null/gammac null mice provide a bioassay system that would detect the functioning of human uNK cells under in vivo conditions.

Problem: TJ6 will be one of the molecules involved in fetal-specific immune suppression during pregnancy. In the mouse and human decidua, the regulation of uterine natural killer (uNK) cells is important during pregnancy., Method of Study: To further understand the possible functions of TJ6 during pregnancy, syngeneic, allogeneic, and mutant mice were examined for TJ6 expression., Results: Immunoblotting showed that TJ6 protein was expressed on most of the placenta-associated mononuclear cells, and the size was 70-72 kDa at all stages of pregnancy. The expression of TJ6 mRNA was studied by a ribonuclease protection assay in syngeneic and allogeneic matings, and in immune-deficient mice of genotypes scid/scid and scid/scid.bg/bg., Conclusions: Genetic disparity, lack of T and B lymphocytes, and loss of NK lytic function had no significant effect on the expression of TJ6 mRNA.

Problem: Stress is known to be abortogenic in animals and humans. An increased decidual release of cytokines such as TNF-alpha and reduction in TGF-beta 2-related immunosuppressive activity has been proposed as the triggering mechanism. Substance P release by nerves in endometrium/decidua has been found to be the key neurotransmitter in this pathway. It is still unclear which cells are stimulated by substance P to produce the increased TNF-alpha level., Method: As a measure of local activation, the granulation of granulated material gland (GMG) cells was measured by flow cytometry after sonic plus immobilization stress of mice or substance P treatment of GMG cells (both isolated GMG cells and GMG-cell containing decidua). TNF-alpha release from decidua and isolated GMG cells was investigated using a TNF-alpha bioassay. The degranulation of uterine mast cell, another potential source of TNF-alpha, was examined in situ by Toluidine blue staining., Results: We observed a striking increase in percentage of degranulated mast cells (8% -->24%) in the uteri of stressed animals, whereas the granularity of GMG cells was decreased by stress but increased with treatment with substance P in vitro. Isolated GMG cells appeared to release in vitro cytotoxins active in the TNF-alpha bioassay, but the magnitude of this activity was not increase by stress or by substance P treatment. In contrast, disaggregated decidual tissue which is known to release increased amounts of TNF-alpha after stress, did increase activity in response to substance P in vitro., Conclusions: Uterine mast cells show activation as reflected by degranulation after stress exposure of pregnant mice and mast cells might be the cellular link between the neurotransmitter substance P and increase in decidual TNF-alpha release that leads to abortion.

Characterisation of murine hybridoma cell lines derived from the fusion of lymphocytes migrating from explant cultures of early, pregnancy-associated metrial glands (days 6-8 of gestation) to SP 2/0 cells, has been extended (van den Heuvel et al., J. Reprod. Immunol., 27 (1994) 13-36). These hybridomas have been grown in culture for over 2 years and are thought to represent the only immortalized lines of murine pregnancy-associated, uterine natural killer (uNK) cells. Previous studies had shown that these hybridomas, known as GWM cells, lack uNK cell surface markers, but share with uNK cells the expression of the lytic protein perforin and the ability to lyse YAC cells, a natural killer cell target (van den Heuvel et al., J. Reprod. Immunol., 27 (1994) 13-36). We report here, the evaluation of the transcription and expression of genes encoding the estrogen receptor (ER), the progesterone receptor (PR) and the interleukin 2 receptor complex (IL 2R alpha, beta and gamma) by uNK cells at day 8 of gestation and by GWM 1-2 cells and SP 2/0 cells. Our investigations indicate that expression of these genes divides day 8 uNK cells into subsets, with the predominant population being ER+, PR-, IL 2R alpha +, IL 2R beta + and IL 2R gamma +. Like day 8 uNK cells, most GWM 1-2 cells expressed all three chains of the IL 2R complex. In addition, GWM 1-2 cells expressed the ER but the PR was not detected on this cell line. Only the IL 2R alpha was detected on the SP 2/0 myeloma cell line. These studies further validate the use of GWM hybridomas as models for pregnancy-associated uNK cells.

Natural killer (NK) cells become a prominent cell population in the rodent uterus during pregnancy. The mature, heavily granulated form of these cells is rare in virgin or postpartum uteri. Death, migration, or dedifferentiation could account for the disappearance of these cells from late gestation uteri. We asked whether uterine NK cells, also known as granulated metrial gland (GMG) cells, die in situ and if expression of Fas antigen is essential for their death. Late in gestation, fragmentation of nuclear DNA was detected histologically by OH-end labeling, as were ultrastructural changes suggesting cell death. NK cells developed in and were lost from the uteri of pregnant Fas antigen-deficient lpr/lpr mice. Postpartum samples of retained placentas contained some residual NK cells that had moved from regions of uterine musculature toward the uterine lumen and were being expelled with the placenta. Thus, both cell death and placental separation remove NK cells from the peripartum uterus.

Granulated metrial gland (GMG) cells are a natural killer (NK) cell-like population present in large numbers in the pregnant rodent uterus. By day 8 of gestation GMG cells are large and granulated and localized to the mesometrial side of each implantation site. GMG cells appear to be highly migratory both in vivo and in vitro; however, little is known regarding their functions. Using indirect fluorescence immunohistochemistry, murine uteri and implantation sites were studied on successive days of gestation to characterize the extracellular matrix receptors of the VLA-integrin family displayed by GMG cells. On days 3 and 6 of gestation, double immunostaining using the monoclonal antibody LGL-1 was employed to recognize GMG cells because their morphology early in pregnancy resembles that of other lymphocytes. Between days 8-15 of gestation, GMG cells can be recognized by their unique morphology. The day 3 and day 6 LGL-1+ cells were positive for all antigens examined; that is, beta 1 plus alpha 1, alpha 3, alpha 4, alpha 5 and alpha 6. From days 8-15 of gestation, GMG cells were beta 1+, alpha 4+, alpha 5+ but alpha 1-, alpha 3-, alpha 6-. Thus, between days 6-8 of gestation, major changes occur in the uterine NK/GMG cell population which include the loss of the surface molecules VLA alpha 1, alpha 3 and alpha 6 or the rapid expansion of NK cells not expressing these proteins. It is postulated that major changes in the functions of uterine NK cells are likely to be associated with these alterations in cell surface phenotype and that functional studies of uterine NK cells should focus upon this relatively early gestational time point.

Seven independent cell lines were derived from the fusion of migratory cells recovered from explant cultures of metrial glands to SP 2/0, a non-Ig secreting B cell myeloma. The migrating cells came from a pool of metrial glands from day 6-8 pregnant random bred CD1 mice and were assumed to be cells early in the differentiation pathway to granulated metrial gland (GMG) cells. The fused cells were cloned twice at the limiting dilution. Hybridization was confirmed by quantitation of cellular DNA using propidium iodide staining and by karyotyping. Electron microscopy revealed that each of the hybrid cell lines was composed of cells which were lymphoid in appearance, but lacked the granules found in mature GMG cells. The surface phenotype of all lines is CD45+, LGL-1-, asialo GM-1-, IgG-, IgM-, CD3- and CD25- (p55 of IL-2 receptor). Although the hybridomas lack those phenotypic markers which were used to show that GMG cells are related to the natural killer (NK) cell lineage (ie LGL-1, asialo GM-1), they do express the pan-leukocyte marker CD45 as well as the lytic protein, perforin, at levels intermediate to those of SP 2/0 cells and GMG cells. In addition, the hybridomas were observed to preferentially bind the NK target cell YAC and to be capable of lytic activity at temperatures below 30 degrees C. Because these hybridomas may represent fusion to an early progenitor cell of the NK/GMG cell lineage, their continued characterization is of merit.

SCID and SCID/beige mice were used to study the pathogenesis of B. catarrhalis administered by intranasal, intraperitoneal or intravenous routes. Challenged adult animals did not appear overtly clinically ill. Similar symptoms were observed regardless of the challenge route, and pretreatment of mice with human transferrin did not enhance clinical virulence. Susceptibility to B. catarrhalis appeared to be age-dependent as some mice under one week of age died following challenge. Postmortem findings included circumscribed pale foci on the liver, splenomegaly and mineralization of the myocardium. Presence of lesions did not correlate with the assessment of clinical well being, and severity of the lesions was found to be challenge strain-dependent. Liver lesions and splenomegaly were not observed in animals challenged with heat-killed bacteria or placebo. SCID/beige mice were more affected than SCID mice both clinically and pathologically, suggesting that natural killer cell and polymorphonuclear cell functions may be important in resolving B. catarrhalis challenge.

The appearance of natural killer (NK) cells during growth and maturation of the murine uterus was studied by immunohistochemistry, using the monoclonal antibody LGL-1. To determine the contributions of microorganisms in the environment and of T-cell and B-cell regulation to the establishment of a uterine NK cell population, uteri from barrier-raised, flora-defined, random-bred CD-1 mice and from genetically T-cell- and B-cell-deficient SCID mice (genotype C.B-17 scid/scid) were compared to uteri from conventionally raised CD-1 mice. Uteri were studied from birth to the ages at which these mice are normally paired for mating (7-10 wk). Absolute uterine weight and the ratio of uterine weight to body weight increased remarkably between 3 and 5 wk of age in each group of animals. Growth continued beyond Week 5 of age, and in all groups the ratio of uterine weight to body weight was similar at puberty, although both the flora-defined CD-1 and SCID mice were significantly smaller than conventionally reared mice. LGL-1+ cells could not be detected in any of the neonatal uteri examined. LGL-1+ cells were first detected at 2 wk of age in uteri from the conventional and flora-defined CD-1 mice. A significant increase in the number of LGL-1+ NK cells occurred in the CD-1 uterus between Weeks 2 and 3 of age and again between Weeks 5 and 7 of age. Environmental conditions did not alter the frequency of LGL-1+ cells between the two groups of CD-1 mice at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Influences of the extracellular matrix (ECM) on the differentiation and distribution of granulated metrial gland (GMG) cells, a uterine natural killer (NK)-like cell subset, were studied by histological examination of implantation sites in the mouse mutant Tsk/+. Tsk/+ mice overproduce collagens I and III. GMG cell differentiation appeared to progress normally in Tsk/+ mice between days 6.5 and 12.5 of gestation. The distribution of GMG cells, however, was abnormal. Significant numbers of GMG cells were found in the antimesometrial and lateral decidual regions at day 8.5 of gestation and in the regions between implantation sites until day 10.5 of gestation. Loss of GMG cells from these regions normally occurs by day 6.5 of gestation. These data suggest that alterations to the ECM change the migration properties or life span of GMG cells.