Jane E. Collins

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. J. Leukoc. Biol. 61: 427–435; 1997.

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.

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 Vβ2 or Vβ8 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 Vβ2 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.

Structure/Function Analysis of Tristetraprolin (TTP): p38 Stress-Activated Protein Kinase and Lipopolysaccharide Stimulation Do Not Alter TTP Function

Tristetraprolin (TTP) is the only trans-acting factor shown to be capable of regulating AU-rich element-dependent mRNA turnover at the level of the intact animal; however, the mechanism by which TTP mediated RNA instability is unknown. Using an established model system, we performed structure/function analysis with TTP as well as examined the current hypothesis that TTP function is regulated by p38-MAPKAP kinase 2 (MK2) activation. Deletion of either the N- or C-terminal domains inhibited TTP function. Extensive mutagenesis, up to 16%, of serines and threonines, some of which were predicted to mediate proteasomal targeting, did not alter human TTP function. Mutation of the conserved MK2 phosphorylation sites enhanced human TTP function in both resting and p38-stress-activated protein kinase-MK2-activated cells. However, p38-stress-activated protein kinase-MK2 activation did not alter the activity of either wild-type or mutant TTP. TTP localized to the stress granules, with arsenite treatment reducing this localization. In contrast, arsenite treatment enhanced stress granule localization of the MK2 mutant, consistent with the involvement of additional pathways regulating this event. Finally, we determined that, in response to LPS stimulation, human TTP moves onto the polysomes, and this movement occurs in the absence of 14-3-3. Taken together, these data indicate that, although p38 activation alters TTP entry into the stress granule, it does not alter TTP function. Moreover, the interaction of TTP with 14-3-3, which may limit entry into the stress granule, is not involved in the downstream message stabilization events.

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.

Autoantibody Responses to Carbohydrate Epitopes in Endometriosis

Abstract: Autoantibody responses to endometrial and serum antigens are a common feature of endometriosis. We have shown that the serum autoantibody response in endometriosis to a number of previously identified antigens, including α2‐Heremans Schmidt glycoprotein and carbonic anhydrase, is specific for a carbohydrate epitope common to these proteins. Removal of carbohydrate moieties from these antigens resulted in a loss of antibody binding. Antibody reactivity was abolished following adsorption with the lectin jacalin, which specifically binds the Thomsen‐Friedenreich (T) antigen (Galβ1‐3GalNAc). Demonstration that the autoantibodies also reacted with other Thomsen‐Friedenreich antigen‐bearing proteins, such as serum IgA1, hemopexin, and MMP‐9, confirmed that this glycotope is involved in the autoantibody response. However, the autoantibody binding requires the presence of at least one sialic acid residue. Thus, the glycotope involved may be a sialylated T antigen. These findings allow us to hypothesize a number of mechanisms whereby the autoimmune response plays a direct role in several aspects of the disease process. The proposed mechanisms take into account the salient endocrine dependency of endometriotic lesions and other aspects of the disease process such as aberrant matrix metalloproteinase function and the ability of endometrial cells to implant at ectopic sites. The anti‐T‐like response may also be indicative of an underlying genetic defect in glycosylation or in the control of glycosylation by steroid sex hormones. Further characterization of this autoimmune response may prove useful in the development of serum‐based diagnostic tests for endometriosis and may lead to the development of therapeutic strategies.

A Subset of Human Uterine Endometrial Macrophages is Alternatively Activated

Human uterine macrophages must maintain an environment hospitable to implantation and pregnancy and simultaneously provide protection against pathogens. Although macrophages comprise a significant portion of leukocytes within the uterine endometrium, the activation profile and functional response of these cells to endotoxin are unknown.

Inhibition of Human Polymorphonuclear Cell Oxidative Burst by 17‐β‐estradiol and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin

Problem:  Polymorphonuclear cell (PMN) function may be directly influenced by 17‐β‐estradiol and the endocrine disruptor, 2,3,7,8‐tetrachloro‐dibenzo‐p‐dioxin (TCDD). This may have significant consequences on PMN function within the female reproductive tract. This study evaluated the effects of 17‐β‐estradiol and TCDD on PMN oxidative burst.

Glucocorticoids enhance the in vivo migratory response of human monocytes.

Glucocorticoids (GCs) are best known for their potent anti-inflammatory effects. However, an emerging model for glucocorticoid (GC) regulation of in vivo inflammation also includes a delayed, preparatory effect that manifests as enhanced inflammation following exposure to an inflammatory stimulus. When GCs are transiently elevated in vivo following exposure to a stressful event, this model proposes that a subsequent period of increased inflammatory responsiveness is adaptive because it enhances resistance to a subsequent stressor. In the present study, we examined the migratory response of human monocytes/macrophages following transient in vivo exposure to stress-associated concentrations of cortisol. Participants were administered cortisol for 6h to elevate in vivo cortisol levels to approximate those observed during major systemic stress. Monocytes in peripheral blood and macrophages in sterile inflammatory tissue (skin blisters) were studied before and after exposure to cortisol or placebo. We found that exposure to cortisol induced transient upregulation of monocyte mRNA for CCR2, the receptor for monocyte chemotactic protein-1 (MCP-1/CCL2) as well as for the chemokine receptor CX3CR1. At the same time, mRNA for the transcription factor IκBα was decreased. Monocyte surface expression of CCR2 but not CX3CR1 increased in the first 24h after cortisol exposure. Transient exposure to cortisol also led to an increased number of macrophages and neutrophils in fluid derived from a sterile inflammatory site in vivo. These findings suggest that the delayed, pro-inflammatory effects of cortisol on the human inflammatory responses may include enhanced localization of effector cells at sites of in vivo inflammation.

A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells

The study of post-transcriptional regulation is constrained by the technical limitations associated with both transient and stable transfection of chimeric reporter plasmids examining the activity of 3′-UTR cis-acting elements. We report the adaptation of a commercially available system that enables consistent stable integration of chimeric reporter cDNA into a single genomic site in which transcription is induced by tetracycline. Using this system, we demonstrate the tight control afforded by this system and its suitability in mapping the regulatory function of defined cis-acting elements in the human TNF 3′-UTR, as well as the distinct effects of serum starvation on transiently transfected and stably integrated chimeric reporter genes.