Earl L. Parr

The Implantation Reaction

The evolution of the reproductive process has resulted in the development of elaborate adaptive mechanisms to ensure the survival of the offspring. In viviparous animals such adaptations include the development of complex and diverse forms of implantation and placentation in order to support the attachment and development of embryos in utero. The implantation process exhibits remarkable diversity among species, most prominently in the extent of trophoblastic invasion into the uterus. Yet the general aim of implantation is accomplished in all species: to attach the embryo to the uterine wall and to establish an intimate union between maternal and fetal tissues so that an exchange of nutrients and waste products can occur. The details of the earliest interactions between the blastocyst and endometrium have been the focus of extensive study. It is our purpose to summarize some of the more important developments in this field since the publication of Finn’s fine review of the subject in the second edition of this book (Finn, 1977). In this chapter we have chosen to discuss four aspects of the implantation process: (1) adhesion of the trophoblast to the uterine epithelium; (2) increased vascular permeability at implantation sites; (3) the decidual cell reaction; and (4) loss of epithelial cells surrounding the implanting blastocyst. Our discussion is based primarily on the results of investigations using common laboratory animals, namely, the rat, mouse, and rabbit. In addition, other recent reviews and symposia may be of interest to the reader (Weitlauf, 1979; Glasser and McCormack, 1981; Finn, 1980; Leroy et al., 1980; Glasser and Bullock, 1981; Dey and Johnson, 1980; Kearns and Lala, 1983; Bell, 1983; Kennedy, 1983a; Chevez, 1984; Enders et al., 1983, 1985; Enders and Schlafke, 1986; Yoshinaga et al., 1986).

SECRETORY IMMUNE RESPONSES IN THE MOUSE VAGINA AFTER PARENTERAL OR INTRAVAGINAL IMMUNIZATION WITH AN IMMUNOSTIMULATING COMPLEX (ISCOM)

Immunostimulating complexes (ISCOMs) are subunit vaccines that are particularly effective in producing immunity against systemic viral infections, but their effectiveness against mucosal infections has received little attention. To study their ability to produce mucosal immune responses in the female reproductive tract, a model ISCOM was prepared containing sheep erythrocyte membrane proteins, and anti-erythrocyte IgA and IgG titres in mouse vaginal washings were measured after immunization at parenteral or local mucosal sites. The ISCOM was prepared by a modified procedure that resulted in incorporation of 10-15% of initial membrane protein compared with 1-5% previously reported. Electrophoretic analysis demonstrated that four out of five erythrocyte membrane proteins were incorporated into the ISCOM, and electron microscopic observations indicated that the ISCOM had a cage-like structure with a diameter of 40 nm, similar to previous ISCOMs. Immunization in the pelvic presacral space (p.s.-p.s.) stimulated significantly higher anti-erythrocyte IgA titres in vaginal fluid than were produced by intraperitoneal (i.p.-i.p.), subcutaneous (s.c.-s.c.), intravaginal (i.vag.-i.vag.), or i.p.-i.vag. immunizations with the same vaccine. Specific IgG titres were less dependent on the route of immunization, with p.s.-p.s., i.p.-i.p. and s.c.-s.c. administration all giving similar high titres while i.p.-i.vag. treatment induced lower titres. These observations using a model ISCOM indicate that mucosal immune responses against membrane proteins were elicited in the female reproductive tract, and that non-mucosal immunization in the pelvis was a more effective route of administration than local application of the ISCOM to the vaginal mucosa.

Antigen recognition in the female reproductive tract. I, Uptake of intraluminal protein tracers in the mouse vagina

Local immunization in the vagina of several species elicits immune responses, but little is known about the uptake, processing and recognition of antigens at this site. We investigated the uptake of intravaginally administered tracers using FITC-bovine albumin, FITC-horse ferritin and FITC-horseradish peroxidase in non-pregnant and pregnant mice. Tracers were detected in cells in the vaginal epithelium and stroma at diestrus, proestrus and metestrus, but not at estrus. During pregnancy, racers were present in vaginal cells on Day 6 but not on Day 13. The distribution of tracers in the vagina was the same in all mice. They were present in vaginal epithelium in cells similar to Langerhans cells and in the stroma in cells that resembled dendritic cells, fibroblasts or macrophages. In some non-pregnant mice, tracers were present in cells adjacent to lymphatic nodules located in the adventitia between the vagina and urethra. Tracers were seen in phagocytic cells lining the marginal and medullary sinuses of the draining lymph nodes (iliac nodes) in some non-pregnant mice at 4 h after intravaginal administration, or in small, dendritic cells in the paracortex at 17 h. To test the possibility that transfer of proteins into the vagina was due to toxic effects of the tracers, FITC-conjugated proteins were also administered into the lumen of uterine horns, and their distribution in horns, cervix and vagina was studied. In uterine horns, tracers were either absent or were located only in apical vesicles in the luminal epithelium. Tracers were present in the cervix and vagina as described above for intravaginal tracers. This result suggests that uptake of tracers in the vagina was not due to toxic effects, and that the vagina and cervix are major sites of protein uptake into the reproductive tract.

VAGINAL IMMUNITY IN THE HSV-2 MOUSE MODEL

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against such infections. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.

Protective immunity against HSV-2 in the mouse vagina

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. The high prevalence of HSV-2 in humans underscores the need to develop an effective vaccine. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against infections in animal models. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.

A comparison of specific antibody responses in mouse vaginal fluid after immunization by several routes

Mice were immunized with a protein antigen, horse ferritin, by eight different routes and the immune responses in the reproductive tract were compared by measuring specific IgA and IgG in vaginal fluid and by localizing anti-ferritin plasma cells in uterine horns, cervix and vagina. The eight routes of immunization were: subcutaneous with Freunds adjuvant (s.c.), intragastric (i.g.), intravaginal (i.v.), s.c.-i.g., s.c.-i.v., i.g.-i.v., i.v.-i.v. and s.c.-i.g.-i.v. The largest overall response, considering both IgA and IgG antibodies, was obtained by s.c. priming with ferritin in adjuvant followed by i.v. boosting. Intravaginal immunization also boosted priming by the i.g., s.c.-i.g. and i.v. routes, but the response to i.v. immunization alone was weak. All i.v. immunizations stimulated mainly IgA antibody responses in vaginal fluid. Specific plasma cells, mostly of the IgG isotype, were present in the vaginal fornix of several mice in the s.c.-i.v. and s.c.-i.g.-i.v. groups, but none were detected there in any other group and they were only rarely observed in the uterine horns. The results provide data on the relative effectiveness of different routes of immunization in producing a humoral immune response in vaginal fluid against a non-replicating antigen.

Immunity to vaginal infection by herpes simplex virus type 2 in adult mice: characterization of the immunoglobulins in vaginal mucus

Progestin-treated female mice are susceptible to vaginal infection by two sexually transmitted disease organisms: herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis. Vaccination of mice with HSV-2 or chlamydial antigens elicits immunity to vaginal infection that may be due in part to secreted antibodies in the vaginal lumen. Analysis of the role of these antibodies in immunity would be aided by information about the vaginal secretion in progestin-treated mice and the antibodies it contains. Gross and histologic observations of progestin-treated mice that were immune to vaginal HSV-2 infection indicated that the vaginal lumen was filled with mucus. A procedure for extraction of immunoglobulin from the mucus was developed and shown to recover at least 98% of the secretory IgA (S-IgA) that was free to diffuse from the mucus. Immunoblotting revealed that the predominant molecular form of IgA in vaginal mucus was dimeric S-IgA. Immunoglobulin concentrations in vaginal secretions were higher in immune mice than in non-immune mice and S-IgA concentrations were higher than those of IgG. The IgG concentration in vaginal secretions of immune mice was 4.5-fold higher than in non-immune mice, while serum IgG increased only 1.5-fold, suggesting local production of IgG or increased transudation in immune mice. Specific IgG antibody to HSV-2 was demonstrated in vaginal secretions of immune mice at a mean ELISA titer of 6200, whereas the titer of specific S-IgA in the same secretions was only 1.9. Thus, while the predominant immunoglobulin by weight in the vaginal mucus of immune mice was S-IgA, the ELISA titers suggested that the virus-specific antibody was almost entirely IgG.

Purification and measurement of secretory IgA in mouse milk

An important factor limiting better understanding of the protective role of sIgA at mucosal surfaces is the limited availability of the purified immunoglobulin. Among other things, purified sIgA is needed for use as a standard in measurements of the concentration of this immunoglobulin in mucosal secretions, particularly in mice, where several models of mucosal infections are available. We describe here a simple method by which one can obtain a mean of 3.5 ml of milk per mouse without a breast pump. Immunoblotting studies after native PAGE demonstrated that the milk contained mainly 420 kDa dimeric sIgA and higher polymeric forms of sIgA; only a trace of monomeric IgA was present. Similar immunoblotting studies after SDS-PAGE revealed that a portion of the sIgA was dissociated by this treatment. The 420 kDa sIgA was purified by salt fractionation, gel filtration, and affinity chromatography, and the purity of the final product was demonstrated by immunoblot analysis of biotinylated polypeptides after reduction of biotinylated protein. The concentration of 420 kDa sIgA in whey was measured by densitometry of immunoblot bands, using the purified 420 kDa sIgA as a standard, and found to be 1.0 +/- 0.3 mg/ml.

Intravaginal Administration of Herpes Simplex Virus Type 2 to Mice Leads to Infection of Several Neural and Extraneural Sites

Female mice have been used extensively to study mucosal immunity against herpes simplex virus type 2 (HSV-2) infection of the vagina, but comparatively little is known about the spread of this virus to other tissues. Here the authors have used immunolabeling to demonstrate that HSV-2 infected the vaginal epithelium; the epithelium covering the vulva, perineum, and anal canal; and perineal hair follicles and sebaceous glands. The kinetics and basal localization of the immunolabeling indicated that the virus spread horizontally within the epithelial layer, starting in the vagina and then proceeding to the distal epithelial sites. HSV-2 also spread from the vagina to multiple neuronal sites including the paracervical ganglia (PCG), which are the major autonomic ganglia of the pelvis. The authors demonstrated both sympathetic and parasympathetic neurons in the PCG by labeling of acetylcholinesterase and tryosine hydroxlyase, and noted that infection was limited mainly or entirely to parasympathetic neurons. Infection of the PCG was correlated with the presence of virus in the autonomic ganglia in the walls of the rectum and urinary bladder, which in turn correlated with distention of these organs and retention of urine and feces. HSV-2 infection was also detected in cell bodies and axons in the lumbosacral sympathetic chain, in lumbosacral dorsal root ganglia, and in the dorsal portions of the lumbar spinal cord. Collectively, the data show that vaginal HSV-2 infection in mice leads to subsequent infection of multiple neural and epithelial sites. This information should be useful for development of a mouse model that can be used to study HSV-2 latency and for development of therapeutic vaccines to prevent recurrent infections.

The effect of adjuvants on antibody titers in mouse vaginal fluid after intravaginal immunization

Intravaginal (ivag) immunization elicits secretory immune responses in the female reproductive tract, but little is known about the safety and effectiveness of adjuvants for such immunization. Mice were immunized intravaginally once daily for 5 days with large doses of horse ferritin combined with aluminum hydroxide (AH), muramyl dipeptide (MDP), monophosphoryl lipid A (MPL), dimethyl dioctadecyl ammonium bromide (DDA) or cholera toxin (CT). Titers of anti-ferritin IgA and IgG were measured in vaginal fluid by ELISA. The most effective adjuvant for ivag primary immunization was AH, while MPL was most effective for ivag boosting. None of the adjuvants caused a detectable tissue reaction in vaginal mucosa. Primary ivag immunization for 5 days with ferritin and AH followed by ivag boosting for 5 days with ferritin and MPL elicited higher IgA titers in vaginal fluid than systemic priming and boosting with ferritin and AH or systemic priming and ivag boosting with ferritin and MPL. Systemically immunized animals exhibited the highest IgG titers in vaginal fluid. The data indicate that adjuvants, particularly AH, can increase local immune responses to intravaginal immunization, but it should be noted that multiple applications of large doses of antigen were used and that this route of sensitization may be relatively inefficient.