Unique CD8+ T cell-rich lymphoid aggregates in human uterine endometrium.

Using confocal scanning laser microscopy of viable tissue sections, we have demonstrated organized lymphoid aggregates (LA), that have a unique structure, in the stratum basalis of uterine endometrium. These LA consist of a core of B cells surrounded by more numerous T cells and an outer halo of monocytes/ macrophages. The T cells in the LA were almost exclusively CD8+CD4-. These CD8+ LA, in terms of both their T cell and B cell components, were either small or absent during the early proliferative stage of the menstrual cycle, significantly larger in size at mid-cycle and during the secretory phase, and absent in post-menopausal women, suggesting that their development is hormonally influenced. This new finding of a menstrual cycle-dependent, phenotypically unique, organized immune cell structure may lead to new insights into the mechanisms by which the endometrium accepts a semiallogeneic graft while providing resistance to infectious organisms.

The mucosal immune system in the human female reproductive tract: potential insights into the heterosexual transmission of HIV.

Using isolated cell suspensions and in situ techniques, we have partially characterized the organization, functional capacity, and sex hormone regulation of the mucosal immune system in the human female reproductive tract. Isolated cells suspensions have been used to demonstrate that the uterus contains antigen-presenting cells that are functionally able to present antigen to autologous tetanus toxoid-specific T cells. Immunophenotypic analyses of the female reproductive tract by three-color immunofluorescent staining has been used to show that lymphoid aggregates, which are absent in postmenopausal women, develop in the uterine endometrium during the menstrual cycle in premenopausal women. Lymphoid aggregates are composed of a B lymphocyte core surrounded by numerous CD8+CD4- T lymphocytes and an outer halo of macrophages. Macrophages, CD4+ and CD8+ T cells, and CD56+ NK cells are distributed throughout the uterine endometrium. In contrast, the Fallopian tube, cervix, and vagina, which lack lymphoid aggregates, contain CD8+ and CD4+ T cells as well as macrophages. The female reproductive tract has also been analyzed for the presence of antigen-independent CD3+ T lymphocyte cytolytic function by an anti-CD3 MAb-mediated redirected lysis assay. High levels of CD3+ T lymphocyte cytolytic activity were demonstrated in cervix and vagina and independent of stage of the menstrual cycle. In the uterus, cytolytic activity changed with endocrine state. In postmenopausal women the uterine endometrium had CD3+ T lymphocytes with high cytolytic activity, whereas premenopausal women had CD3+ T lymphocytes with moderate cytolytic potential during the proliferative phase to low/no cytolytic activity during the secretory phase of the menstrual cycle. In studies to determine whether the upper reproductive tract could be infected with HIV-1, we found on the basis of nef expression and p24 release that epithelial cells from the Fallopian tube, and from the uterus and cervix, are infectable. These studies demonstrate that the human female reproductive tract is an inductive site for immune responses and the cell-mediated immunity is present throughout the female reproductive tract. These studies further indicate that the Fallopian tube and uterus are potential entry sites for HIV-1 infection and that uterine immune cell architecture as well as cytolytic activity are under hormonal control.

MHC class II expression and antigen presentation by human endometrial cells.

It has been demonstrated previously that mixed cell suspensions from the female reproductive tract consisting of human epithelial and stromal cells were capable of presenting foreign antigen to autologous T cells. There have been, however, no reported studies examining antigen presentation by isolated epithelial cells from the human female reproductive tract. It is now shown that freshly isolated epithelial cells from the uterine endometrium constitutively express MHC class II antigen and that class II was upregulated on cultured epithelium by interferon gamma (IFNgamma). Using a highly purified preparation, it was demonstrated that these epithelial cells were able to process and present tetanus toxoid recall antigen driving autologous T cell proliferation. Cells isolated from the basolateral sub-epithelium stroma were also potent antigen presenting cells in this model system. Thus, isolated endometrial epithelial cells were able to directly process and present antigen to T cells and may be responsible for the transcytosis and delivery of antigen to professional antigen presenting cells found in the sub-epithelial stroma.

Association of IgA-Fc receptors (Fc alpha R) with Fc epsilon RI gamma 2 subunits in U937 cells. Aggregation induces the tyrosine phosphorylation of gamma 2.

We investigated the possibility that IgA-binding chains of Fc alpha R on monocytic cells are physically associated with gamma 2 subunits of Fc epsilon RI (Fc epsilon RI gamma 2 or gamma 2). Fc alpha R was precipitated from lysates of IFN gamma-treated U937 cells, subclone 10.6, and probed by immunoblotting with Ab against human gamma 2. Fc alpha R was precipitated through anti-Fc alpha R mAbs A59 or A62, through A62 from lysates that had been exhaustively precleared of high affinity IgG-Fc receptors (Fc gamma RI) and of low affinity Fc gamma RII, and through anti-Fc alpha R mAb A77 from Fc gamma RI-precleared lysates of untreated 10.6 cells. precipitation was also performed through F(ab')2 A77 and through the native ligand of the receptor, hlgA. In all cases, Fc alpha R precipitates contained co-isolated 22-kDa gamma 2 (unreduced). The Fc alpha R alpha-chain/gamma 2 complex did not readily dissociate in 1% Nonidet P-40 as did Fc gamma RI alpha-chain/gamma 2, suggesting a novel aspect to the Fc alpha R subunit interaction. Specific Fc alpha R aggregation on cells triggered a robust respiratory burst and the tyrosine phosphorylation of several proteins. Among them was phospho-gamma 2, which migrated as a 24- to 28-kDa gamma 2 phosphoprotein on gels and was detected as a 28-kDa phosphoprotein by anti-phosphotyrosine immunoblot. Aggregated Fc alpha Rs that were precipitated from Fc alpha R-triggered cells also contained a phosphoprotein of the same mobility and immunoreactivity, as did aggregated Fc gamma RI from which the 28-kDa phosphoprotein could be more readily eluted and identified (as phospho-gamma 2). We concluded that myelocytic Fc alpha Rs are multichain complexes containing gamma 2 subunits that are tyrosine phosphorylated upon Fc alpha R aggregation. As IgA is the predominant Ig on mucosal surfaces, gamma-subunits may play an important role in mucosal immunity involving leukocytic Fc alpha R.

Autoantibodies in endometriosis sera recognize a Thomsen-Friedenreich-like carbohydrate antigen.

Autoantibody responses to endometrial antigens are a common feature of endometriosis. Antibody responses to a number of serum and tissue antigens such as alpha(2)-Heremans Schmidt glycoprotein (alpha(2)-HSG), transferrin, and carbonic anhydrase have been identified. The nature of the epitopes recognized on these proteins has not been determined. In this study we show that the serum antibody response to alpha(2)-HSG and carbonic anhydrase is against a common carbohydrate epitope which is also expressed on bovine fetuin. Removal of carbohydrate moieties from these antigens resulted in loss of antibody binding. Antibody reactivity with alpha(2)-HSG, fetuin and other antigens was removed by binding with the lectin jacalin. Jacalin specifically binds the Thomsen-Friedenreich antigen (Galbeta1-3GalNAc). Demonstrating that the autoantibodies also reacted with other Thomsen-Friedenreich antigen-bearing proteins, serum IgA1 and haemopexin confirmed an association with this epitope. These antigens have not been previously described as autoantigens in endometriosis and are of interest since they raise the possibility that this autoimmune response may either play a direct role in the disease process or reflect an abnormality of glycosylation in endometriosis. These results may also prove useful in the development of a serum diagnostic test for endometriosis.

Opsonization of yeast by human serum IgA anti-mannan antibodies and phagocytosis by human polymorphonuclear leucocytes.

The ability of sera from 72 patients with liver disease to opsonize yeast for phagocytosis by normal polymorphonuclear leucocytes has been studied. Seven showed defective opsonization. The opsonic activity of all but two sera was decreased markedly by heating at 56 degrees C for 1 h. When the two sera with heat stable opsonic activity were fractionated by gel filtration and by ion exchange chromatography, the activity copurified with IgA, not with IgG. The purified IgA, radiolabelled with 125I was shown to bind in a saturable manner to the yeast. Both sera had high levels of anti-yeast mannan IgA detected by an ELISA. In one case most of the anti-mannan activity was due to monomeric IgA, in the other it was dimeric. This was consistent with the observation of an apparent molecular weight of the opsonin of approximately 180 kD in one serum and 300-400 kD in the other.

Immune aspects of endometriosis: relevance of the uterine mucosal immune system.

Endometriosis is classically defined as the growth of endometrial cells at sites outside the uterus. It is a common disease characterized by infertility, chronic pain and adhesion formation. Immune dysregulation, evidenced by decreased clearance of endometrial cells and aberrant production of cytokines by peritoneal fluid leukocytes, has been proposed as a mechanism which allows implantation and growth of ectopic endometrium. Cytokines are primary components of intercellular signaling between uterine epithelial and stromal cells, leukocytes, and the developing conceptus. Because their production is regulated by sex hormones, cytokines are well-placed to play a key role in the extensive tissue remodeling required to accommodate menstruation, implantation and pregnancy. Understanding this specialized hormonally-responsive mucosal immune system within the uterus will be critical to understanding the potential importance of the immune system in the pathogenesis of endometriosis. In this review, highlights of studies describing leukocyte populations, cytokines and cytokine receptors in uterine and ectopic endometrium and their proposed role in the regulation of immune processes and endometrial growth are presented, followed by a review of current data on immune aspects of endometriosis. Studies directed at investigating the hormonal regulation of cytokine secretion by uterine and peritoneal cell populations, and the effect of cytokines on endometrial proliferation, should provide a more complete understanding of their potential role in normal uterine growth and in the pathogenesis of endometriosis.

Characterization of the IgA receptor from human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes (PMNs) will phagocytose yeasts opsonized with specific affinity-purified human serum IgA. PMNs also bind to Sepharose beads coated with IgA or IgG, but not to beads coated with bovine serum albumin (BSA) or horseradish peroxidase (HRP). Binding to IgA-Sepharose stimulates the cells to release lysozyme. Affinity chromatography of 125I-labelled PMN membrane proteins on IgA-Sepharose results in isolation of a polypeptide of apparent 60,000 MW. The protein, which is not bound to IgG-Sepharose under the same conditions, appears as a diffuse band on SDS-PAGE, suggesting it is heavily glycosylated.

Tissue fixation with phenol-formaldehyde for routine histopathology.

The addition of 2% phenol had a marked accelerating effect on neutral buffered 4% formaldehyde as a fixative. Histopathological material fixed in buffered phenol-formaldehyde (pH 7.0) and rapidly advanced to paraffin in an enclosed tissue-processor showed improved nuclear and cytoplasmic detail, reduced shrinkage and distortion, and an absence of formalin pigment. Good results were obtained in less time when sequential fixation in phenol-formaldehyde buffered to pH 7.0 and pH 5.5 was carried out at an elevated temperature (40 degrees C) in the enclosed tissue-processor. Standard histological stains and immunoperoxidase methods worked well. In resin-embedded tissue, buffered phenol-formaldehyde (pH 7.0) gave satisfactory ultrastructural results. The penetration rate of buffered phenol-formaldehyde (pH 7.0) in gelatin models did not differ from that of neutral buffered 4% formaldehyde. Polyacrylamide gel electrophoresis showed enhanced protein polymer formation with buffered phenol-formaldehyde (pH 7.0) as compared with neutral buffered 4% formaldehyde. Protein polymer formation increased in response to increased time and temperature. Cells fixed in suspension in buffered phenol-formaldehyde (pH 7.0) and neutral buffered 4% formaldehyde showed similar volume changes.

Discoid lupus erythematosus in an X-linked cytochrome-positive carrier of chronic granulomatous disease.

A 13-year-old female presented with photosensitivity, recurrent aphthous ulcers and discoid lupus erythematosus (DLE)-like skin lesions. These symptoms have been linked to the carrier status of chronic granulomatous disease (CGD). Neutrophil (PMN) function was investigated by nitroblue tetrazolium reduction test and chemiluminescence. A severe impairment of PMN oxidative burst activity was revealed in spite of supranormal levels of cytochrome b245. Glucose-6-phosphate dehydrogenase activity was deficient. Her mother and two sisters also showed reduced PMN function. These findings are consistent with a cytochrome positive X-linked form of CGD with variable lyonization. DLE in association with the carrier status of this CGD variant has not been reported previously.

Mucosal immunity in the human female reproductive tract: cytotoxic T lymphocyte function in the cervix and vagina of premenopausal and postmenopausal women.

PROBLEM: To investigate the mucosal immune system in the cervix and vagina of premenopausal women in terms of immune cells present and cytolytic capacity of mucosal CD3+ T cells in the lower reproductive tract. METHODS: Fresh tissue fragments prepared by vibratome sectioning were analyzed for the presence of cells by confocal scanning laser microscopy (CSLM). Isolated reproductive tract cells prepared by enzymatic were analyzed for CD3+ T cell phenotype by FACS analysis and for cytolytic function by an anti-CD3 mAb mediated redirected lysis assay. RESULTS: As determined by CSLM, CD3+ cells as well as macrophages and dendritic cells are distributed throughout the lower female reproductive tract in both the epithelium and subepithelial mucosa. It was found that cervical and vaginal tissues from pre- and post-menopausal women contain CD3+ T cells (CTL) that have cytolytic activity, when measured in an antigen non-specific anti-CD3 mAb mediated redirected lysis assay. CONCLUSIONS: These results indicate that the lower reproductive tract of women is immuno-competent as judged by the presence of CD3, CD4, CD8, macrophage, and dendritic cells in the endocervix, ectocervix, and vagina of premenopausal and postmenopausal women. Further, these studies demonstrate that CD3+ T cells with cytolytic activity are present in the cervix and vagina during the proliferative and secretory phases of the menstrual cycle and following menopause.

CD8+ T cells in human uterine endometrial lymphoid aggregates: evidence for accumulation of cells by trafficking.

Lymphoid aggregates (LA) develop during the proliferative phase of the menstrual cycle in the human uterine endometrium (EM). They contain mostly CD8+ T cells and B cells. As these LA are absent immediately following menses, they may arise by division of cells resident in the EM, or by division of a limited number of precursor cells that traffic into the EM during the early proliferative phase of the menstrual cycle. Alternatively, they may arise by the continuous trafficking of cells into the EM throughout the proliferative phase of the menstrual cycle. In this study we investigated the distribution and frequency of CD8+ T cells in the aggregates using expression of Vbeta2 or Vbeta8 as markers of clonality and Ki-67 as a marker of dividing cells. Confocal microscopic analysis of endometrial tissues showed the random distribution of CD8+ T cells within aggregates within the same sample and in aggregates from different samples. Furthermore, comparisons of the distribution of Vbeta2 and Vb8 with expected values predicted from Poisson distribution values were not significantly different, suggesting that CD8+ T cells do not arise by division from single precursors. A low level of T-cell division within LAs was confirmed by positive staining for Ki-67. Dividing T cells were randomly dispersed throughout the LA and the frequency of dividing cells did not vary greatly between aggregates within the same tissue. Nearest-neighbour analysis of dividing cells showed no statistically significant deviations from a random distribution. Taken together, these results suggest that LA develop during the menstrual cycle largely by the trafficking of cells to nucleation sites within the EM, rather than by division of a limited number of precursor cells.

IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils.

Cytokines present in the human uterus play an important role both in modulating immune responses to infectious challenge and in the establishment and maintenance of pregnancy. In particular, successful implantation and pregnancy is thought to require the establishment of a Th2 environment, while Th1 cytokines are associated with pregnancy loss and infertility. On the other hand, a Th1 response appears to be required for the resolution of acute infection. Using novel confocal microscopic analysis of fresh sections of human tissue, we have investigated the production of IFN-gamma, a Th1 cytokine, in human endometria. Extracellular IFN-gamma, mostly associated with matrix components, was located immediately beneath the luminal epithelium and along the glandular epithelium proximal to the lumen. As evidenced by intracellular staining, IFN-gamma is produced by both stromal cells and intraepithelial lymphocytes through all stages of the menstrual cycle. Surprisingly, the stromal cell containing intracellular IFN-gamma was identified as a polymorphonuclear neutrophil on the basis of its reactivity with a panel of mAbs and its nuclear morphology. We further found that polymorphonuclear neutrophils isolated from normal donors produce IFN-gamma in response to stimulation with LPS, IL-12, and TNF-alpha. Taken together, these findings suggest that polymorphonuclear neutrophils are capable of producing IFN-gamma both in vitro and in vivo, indicating that their role in shaping immune responses may be more extensive than previously thought. Furthermore, these studies strongly suggest that polymorphonuclear neutrophils play an important role in determining immune responsiveness within the female reproductive tract.

Mice lacking the chemokine receptor CCR1 show increased susceptibility to Toxoplasma gondii infection.

Chemokines are critical for the recruitment of effector immune cells to sites of infection. Mice lacking the chemokine receptor CCR1 have defects in neutrophil trafficking and proliferation. In the present study, we tested the susceptibility of CCR1 knockout mice to infection with the obligate intracellular protozoan parasite Toxoplasma gondii. In comparison with parental wild-type mice, CCR1(-/-) mice exhibited dramatically increased mortality to T. gondii in association with an increased tissue parasite load. No differences were observed in Ag-specific T cell proliferation or in cytokine responses between mutant and wild-type mice. However, the influx of PMNs to the peripheral blood and to the liver were reduced in CCR1(-/-) mice during early infection. Our results suggest that CCR1-dependent migration of neutrophils to the blood and tissues may have a significant impact in controlling parasite replication.

Human immunodeficiency virus type 1 infection of cells and tissues from the upper and lower human female reproductive tract.

Viable tissue sections and isolated cell cultures from the human fallopian tube, uterus, cervix, and vaginal mucosa were examined for susceptibility to infection with human immunodeficiency virus type 1 (HIV-1). We examined infectivity by using the monocytotropic strain HIV-1(JR-FL) and several primary isolates of HIV-1 obtained from infected neonates. HIV-1 infection was measured by p24 production in short-term culture and by immunofluorescence detection of HIV-1 Nef and p24 proteins by laser scanning confocal microscopy. Three-color immunofluorescence was used to phenotype HIV-infected cells within tissue sections from each site. Our findings indicate that epithelial, stromal, and dendritic cells and cells with CD14+ CD4+, CD14-CD4-, and CD4+ CD14- phenotypes from the female reproductive tract are infectable with HIV-1. Of importance is the finding that tissues from the upper reproductive tract are susceptible to infection with HIV-1. Moreover, tissue samples from women in all stages of the menstrual cycle, including postmenopausal women (inactive), could be infected with HIV-1. Female reproductive tract cells required a minimum of 60 min of exposure to HIV-1 in order for infection to occur, in contrast to peripheral blood lymphocytes, which became infected after being exposed to HIV-1 for only 1 min. These findings demonstrate that HIV-1 can infect cells and tissues from different sites within the female reproductive tract and suggest that multiple cell types, including epithelial cells, may be targets for the initial infection by HIV-1.

Fas-FasL interaction involved in pathogenesis of ocular toxoplasmosis in mice.

Ocular toxoplasmosis is a potentially blinding intraocular inflammation. The intent of this study was to investigate the role of Fas-FasL interaction in a murine model of acquired ocular toxoplasmosis induced by intracameral inoculation of Toxoplasma gondii. Intraocular inflammation, Fas and FasL expression on lymphocytes and on ocular tissues, the occurrence of apoptosis, and the frequency of CD8(+) and CD4(+) T cells in the infected eyes were analyzed in C57BL/6 (B6) mice. Susceptibility to parasite-induced intraocular inflammation was observed in Fas-deficient (B6-lpr) and FasL-deficient (B6-gld) mice. Inoculation of 5,000 T. gondii tachyzoites induced significant intraocular inflammation associated with increase of Fas and FasL expression in the inoculated eyes of wild-type B6 mice. Flow cytometry demonstrated a significant increase of Fas and FasL expression on the splenocytes from naive mice incubated in vitro with the parasite and on the splenocytes harvested from the infected mice at day 8 after parasite inoculation. Apoptosis of inflammatory cells and cells in ocular tissues was seen, and a greater frequency of CD8(+) than CD4(+) T cells was observed in the infected eyes. The intensity of intraocular inflammation was greater in B6-lpr and B6-gld mice than in wild-type B6 mice (P < 0.05). The results suggest that Fas-FasL interaction associated with apoptosis is involved in the pathogenesis of acquired ocular toxoplasmosis in mice.

Human immunodeficiency virus-specific and CD3-redirected cytotoxic T lymphocyte activity in the human female reproductive tract: lack of correlation between mucosa and peripheral blood.

CD8(+) T cell phenotype and function were assessed in the female reproductive tracts (FRTs) of 3 human immunodeficiency virus (HIV)-positive patients who had undergone hysterectomy. FRT cytotoxic T lymphocyte (CTL) lytic activity from 1 patient (patient 872) was detected by using CD3-dependent redirected-lysis assay and HIV-specific assay, concomitant with the presence of CD8(+) cells. In contrast, samples from the 2 other HIV-positive patients (patients 1356 and 1364), who also were asymptomatic for HIV-associated illnesses, demonstrated no CTL activity in any solid tissue tested by either assay, despite activity by autologous peripheral blood mononuclear cells (PBMC). This absence of CTL activity was correlated with a relative absence of CD8(+) cells in the FRT, whereas CD8(+) cells were present in PBMC. Thus, CTL activity in PBMC may fail to correlate with mucosal activity. The finding of CTL activity in the FRT of patient 872 represents the first description of CTL in upper and lower FRT tissues of an HIV-positive woman.