Human beta-defensin-1: an antimicrobial peptide of urogenital tissues.

Antimicrobial peptides are widely distributed mediators of innate host defense in animals and plants. A 36 amino acid antimicrobial peptide belonging to the defensin family, and named human beta-defensin-1 (HBD-1), was purified recently from hemodialysate fluid, but its tissue sources were not identified. By Northern blotting, we found the highest concentrations of HBD-1 mRNA in the kidney and the female reproductive tract. In situ hybridization localized the HBD-1 mRNA in the epithelial layers of the loops of Henle, distal tubules, and the collecting ducts of the kidney and the epithelial layers of the vagina, ectocervix, endocervix, uterus, and fallopian tubes in the female reproductive tract. Using a novel technique designed to detect cationic peptides in urine, we recovered several forms of HBD-1 ranging in length from 36 to 47 amino acid (aa) residues and differing from each other by amino terminal truncation. The total concentration of HBD-1 forms in voided urine was estimated at 10-100 microg/liter, with individual variations in the total amount of HBD-1 peptides and the relative proportion of HBD-1 forms. Multiple forms of HBD-1 (size 36-47 aa) were also found in the blood plasma, bound to carrier macromolecules that released the peptide under acid conditions, and in vaginal mucosal secretions (39, 40, and 44 aa). By immunostaining, HBD-1 was located in the kidney within the lumen of the loops of Henle, but no intracellular storage sites were identified in renal or female reproductive tissues. Recombinant HBD-1 forms (36, 39, and 42 aa) and natural HBD-1 forms were antimicrobial to laboratory and clinical strains of Escherichia coli at micromolar concentrations. HBD-1 activity was not changed appreciably by low pH, but was inhibited by high salt conditions. Some of the HBD-1 peptides retained their activity against E. coli in unconcentrated (low conductance) urine, and the 36 aa form was microbicidal even in normal (high conductance) urine. Production of HBD-1 in the urogenital tract could contribute to local antimicrobial defense.

Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis.

Chlamydia species infect epithelial cells at mucosal surfaces, and are major causes of sexually transmitted diseases. Infection is characterized by inflammation which is exacerbated upon reinfection, ultimately leading to tissue damage and scarring. Although central for the development of disease manifestations, little is known about the mechanisms that initiate and sustain the inflammatory response to Chlamydia. Infection of cervical and colonic epithelial cells with Chlamydia trachomatis and Chlamydia psittaci is shown in the present studies to upregulate mRNA expression and secretion of the proinflammatory cytokines IL-8, GRO alpha, GM-CSF, and IL-6. In contrast to the rapid, but transient, cytokine induction following infection with other invasive bacteria, the epithelial cytokine response to Chlamydia was delayed until 20-24 h after infection, persisted throughout the chlamydial growth cycle (2-4 d), and required bacterial protein synthesis. Moreover, epithelial cell lines and primary endocervical epithelial cells released IL-1alpha after Chlamydia infection, and increased secretion of the proinflammatory cytokines could be inhibited by anti-IL-1alpha. This suggests that IL-1alpha, released following lysis of infected epithelial cells, may amplify the inflammatory response by stimulating additional cytokine production by noninfected neighboring cells. These findings suggest a novel pathophysiologic concept wherein the acute host response to Chlamydia at mucosal surfaces is primarily initiated and sustained by epithelial cells, the first and major targets of chlamydial infection.

The innate and early immune response to pathogen challenge in the female genital tract and the pivotal role of epithelial cells.

The female reproductive tract is immunologically unique in its requirement for tolerance to allogeneic sperm and, in the upper tract, to the conceptus. However, it must also be appropriately protected from, and respond to, a diverse array of sexually transmitted pathogens. Some of these infections can be lethal (e.g. Human Immunodeficiency Virus (HIV), Human Papilloma Virus (HPV)), and others (e.g. Chlamydia trachomatis and Neisseria gonorrhoeae) can have potentially devastating reproductive sequelae. Interactions between a host and a pathogen are complex, diverse and regulated, and are a function of the individual pathogen, and host immunity. Although there is undoubtedly commonality in the mucosal immune response, there is also evidence of a degree of site-specificity in immune mechanisms, dependent upon the function and anatomical location of an organ. In this article, we review the evidence on the pivotal role of epithelial cells in the innate and early immune response to pathogen challenge in female genital tract tissues, and examine the evidence that the 'sterile' upper and the 'non-sterile' lower female genital tract may maintain a different immunological surveillance milieu, and may also respond differentially to pathogen challenge. We also review the unique characteristics, and subsequent ramifications of the acute cervical immune response to C. trachomatis, and discuss how natural antimicrobial mediators of immunity may be utilized to decrease the spread of sexually transmitted infections.

Human seminal plasma suppresses lymphocyte responses in vitro in serum-free medium.

The in vitro immunosuppressive properties of human seminal plasma have been re-investigated in serum-free medium in view of recent suggestions that the previously observed effects might be dependent on the presence of exogenous serum co-factors present in the culture media. The present studies reveal that low concentrations of seminal plasma can inhibit the ability of peripheral blood leukocytes to lyse K562 target cells in the absence of fetal calf or new-born calf serum. These inhibitory effects could be achieved by pre-incubating the effector cells in seminal plasma at 37 degrees C prior to use in the natural killer cell assay or by incorporating it into the assay system. Additional studies revealed that human seminal plasma could also inhibit the proliferative response of peripheral blood lymphocytes to phytohaemagglutinin in serum-free HB103 medium. These effects were most marked and consistent if the seminal plasma was present throughout the period of culture. Overall, these studies indicate that the previously reported suppressive effects of human seminal plasma in these systems cannot be entirely attributable to cytotoxic factors generated by exogenous serum components.

The case against an association between HIV-1 and sperm: molecular evidence.

In this article we present data from our laboratory, and review the literature available, on the potential association between HIV-1 and sperm. We focus on the use of PCR technology to answer this very important question, and emphasise the importance of using highly purified sperm preparations. We conclude that the likelihood of HIV infection/association with viable mature sperm is low.

Immunosuppression by seminal plasma from fertile and infertile men: inhibition of natural killer cell function correlates with seminal PG concentration.

Human seminal plasma has uniquely high concentrations of PGE and 19-hydroxy PGE but the function of these PGs has not been elucidated. PGs of the E series have been shown to be paracrine and autocrine regulators of the function of immune cells and high levels of PGE have been shown consistently to suppress function in such cells. Human seminal plasma has a potent immunosuppressive effect and evidence is accumulating that this is largely due to PG components. In this study the effects of human seminal plasma on the killing activity of natural killer (NK) cells as judged by 51Cr release from K562 cells have been studied in groups of fertile and infertile men. Although there was no significant difference in the PGE, 19-hydroxy PGE or the NK cell inhibitory activity in the two groups, the inhibition of NK cell activity was closely correlated with the PGE and the 19-OH PGE content of the seminal plasma in the fertile group. This finding is further evidence that the major contribution to the immunosuppressive properties of human semen is provide by the high concentration of PGs of the E series in this fluid.

Immunosuppression by seminal plasma and its possible biological significance.

The data support the role of seminal plasma facilitating the transmission of virus and establishment of infection when all depositions of semen occur, but systemic absorption would also be possible by vaginal intercourse if lesions or abrasions caused for example by other s.t.d's, were present. The severity and rapid increase in the population of certain sexually transmitted agents make it imperative that epidemiological studies are initiated to study any correlation with changes in sexual behaviour and contraceptive practices. Finally, further isolation and characterization of seminal plasma components should lead to a clear understanding of their action as both regulatory molecules and in their possible contribution to disease.

The psychology of briefing. A review and seven recommendations for improving performance.

In many operational settings, briefings take place at the beginning of a work period. Often, such briefings are made without much forethought and training. This article outlines some of the problems that are likely to be encountered in such situations and provides seven recommendations that should improve the memorability and comprehensibility of briefing sessions. It is recommended that briefings should begin with an outline of the critical points, be devoid of unnecessary detail and be confined to not more than six topics. Guidelines are provided for the use of pictures and graphs and combining these with verbal descriptions. The value of handouts and the use of summaries and prompts at the end of the briefing session are also considered.

Updating one's knowledge about the current status of vehicles in a freight system simulation.

The use of simulations of real-world settings can provide data relevant to ergonomics problems in a wide range of similar settings. This paper reports on a simulation of a freight service operation. It examines the effect of manipulating the type and format of information representation on the ability to update knowledge about the status of three vehicles. In addition, the effects of display type and the detection of violations of pre-learned rules on the ability to update are examined. The display format proved not to be a critical factor but the requirement to detect rule violations of a specific vehicle resulted in better memory-updating performance for that vehicle. The results are discussed in terms of a human memory mechanism, which is analogous to a computer operating system, involved in updating memory and detecting violations. Finally, the value of using this tool to examine complex cognitive processes relevant to the workplace is discussed.

Characterization of T lymphocytes and antigen-presenting cells in the murine male urethra.

The male lower urogenital tract is exposed to sexually transmitted pathogens and is therefore a strategic site of immune defense. To further define the immunodynamics of this region, we studied the histology, immune cell distribution, and draining lymph nodes of the murine male lower urogenital tract. The external surface of the foreskin was covered by skin composed of keratinized stratified epithelium containing numerous hair follicles and sebaceous glands. Immunologically the penile foreskin was characterized by the presence of few T lymphocytes and macrophages. Numerous Langerhans cells, however, were detected within the epithelium. The penile urethra was composed of stratified columnar epithelium, with a meatus lined by keratinized squamous epithelium preceding the opening proper. The most abundant immune cells of the penile urethra were macrophages. In young adult, virgin males, these were found primarily underlying the urethral epithelium, but in older, mated mice, they were usually intraepithelial in location, and were more abundant. Langerhans cells could not be specifically identified in the urethral mucosa. T lymphocytes were found underlying and occasionally within the epithelium of the urethral mucosa, with CD4+ cells more abundant than CD8+ cells. The majority of lymphocytes observed around the urethra were positive for the integrin beta 7 alpha M290, which is selectively expressed by mucosal lymphocytes, providing indirect evidence that the urethra is part of the common mucosal system. Lymphocytes expressing the gamma delta T cell receptor and IgA-positive plasma cells were not detected. The primary draining nodes for the vas deferens and urethra were the lumbar nodes. Lymphatic drainage from the rectum also involved the lumbar nodes. Information obtained in this study should help to elucidate optimal genital tract vaccination strategies for defense of the male urogenital tract against sexually transmitted pathogens.

Different cytokine production profiles of gamma delta T cell clones: relation to inflammatory arthritis.

This study was performed to investigate whether gamma delta T cells could also be divided into subsets, identified by a cytokine profile, as described for alpha beta T helper (Th) cell subsets. Cytokine production was studied in 22 gamma delta T cell clones obtained from the synovial fluid and peripheral blood of one patient with inflammatory arthritis and compared to that of 26 alpha beta T cell clones of the same and different patients. Interferon-gamma (IFN-gamma) was produced by 18 (82%) and interleukin-4 (IL-4) by 17 (77%) out of 22 gamma delta T cell clones, respectively. In contrast, IL-10 was not produced, except at very low level in one case. The mean levels of IL-4 were lower for clones derived from synovial fluid. When considering the production of IFN-gamma as an indicator of Th1 and that of IL-4 as an indicator of Th2, respectively, the most common pattern was a gamma delta Th1-like pattern, with the combination of high levels of IFN-gamma and low levels of IL-4. This pattern was found in V delta 1+ clones, all from synovial fluid. Additional patterns were also observed: a mixed, probably gamma delta Th0-like pattern with a more balanced production of both IFN-gamma and IL-4; a gamma delta Th1 pattern with the production of IFN-gamma alone; a gamma delta Th2 pattern with the production of IL-4 alone. These three patterns were also seen in blood gamma delta T cells which were all V delta 2, indicating that these patterns were independent of the V delta phenotype. gamma delta T cell clones produced lower levels of IFN-gamma (p = 0.001) and higher levels of IL-4 than alpha beta clones (p < 0.02). These differences in cytokine production between alpha beta and gamma delta subsets and within these subsets may contribute to their respective role in chronic inflammation.

Studies on the immunosuppressive effect of seminal plasma.

In vitro suppression of immune responses by seminal plasma is well documented, but the mechanism by which it exerts its effects remains to be established. Our studies on T-lymphocyte proliferation and natural killer cell target-cell lysis reveal that seminal plasma mediated suppression is dose-dependent and temperature-dependent, and that cells which have been activated are less susceptible to suppression. In the case of mitogen-induced T-cell responses this results in a decrease in the expression of the Interleukin-2 receptor whose generation is essential to T-cell proliferation. These studies provide further evidence about suppression of the immune response by seminal plasma. This may be a contributory factor in the aetiology of AIDS, other sexually transmitted diseases, infertility and malignancies of the urogenital tract including carcinoma of the cervix.

Selective activation of resting human gamma delta T lymphocytes by interleukin-2.

In rheumatoid arthritis and other inflammatory diseases we and others have found that gamma delta T cells express activation antigens, suggesting that they are involved in the pathogenesis of these disorders. In this study we have stimulated peripheral blood mononuclear cells from normal donors with recombinant interleukin-2 (rIL-2) to see whether such a stimulus alone could activate gamma delta T cells. Short-term exposure (24-96 h) to rIL-2 selectively stimulated the gamma delta but not the alpha beta T cells to express activation antigens (CD69, CD25 and HLA-DR). Long-term culture (2 weeks) in rIL-2-containing medium caused a selective increase in the proportion of the gamma delta T cells and a corresponding reduction of the fraction of alpha beta T cells. Limiting dilution analysis revealed that approximately 1/60 of the gamma delta T cells responded to IL-2 in contrast to only 1/250 of the alpha beta T cells. Comparison of the expression of the IL-2 receptor (IL-2R) alpha and beta chains showed that there was a similar expression of the alpha chain on gamma delta and alpha beta T cells whereas the relative density of the beta chain was more than twice as high on gamma delta T cells. Both the IL-2-induced proliferation of gamma delta T cells and the expression of activation antigens on these cells could be inhibited by an anti-IL-2R beta monoclonal antibody (mAb) but not by an anti-IL-2R alpha mAb. Expression of CD69 on gamma delta T cells was dependent neither on the presence of B cells, monocytes, nor alpha beta T cells. Finally, we found that the IL-2-induced expression of CD69 was inhibited by activation of cAMP-dependent protein kinase and by inhibition of the Src-family of the tyrosine protein kinase, but not by inhibition of protein kinase C or by activation of the CD45 associated tyrosine phosphatase. The ability of gamma delta T cells to be activated by IL-2 is a feature which they have in common with natural killer cells. Moreover, it may be possible that the expression of activation antigens on gamma delta T cells in inflammatory diseases is an epiphenomenon secondary to IL-2 produced by activated alpha beta T cells.

A high proportion of the V delta 1+ synovial fluid gamma delta T cells in juvenile rheumatoid arthritis patients express the very early activation marker CD69, but carry the high molecular weight isoform of the leucocyte common antigen (CD45RA).

We have previously shown that gamma delta T cells in the synovial compartment of patients with juvenile rheumatoid arthritis (JRA) express activation antigens (CD69 and HLA-DR) and that they are predominantly of the V delta 1 subset. In this study we have analysed the expression of activation antigens (CD69 and HLA-DR) and different isoforms of the leucocyte common antigen (CD45RO and CD45RA) on the V delta 1 and the V delta 2 subsets of gamma delta T cells in paired samples of synovial fluid and peripheral blood of nine patients with JRA, and in the peripheral blood of five children with idiopathic scoliosis. In the synovial fluid of children with JRA, there were significantly more V delta 1+CD69+ and V delta 2+CD69+ cells compared with the peripheral blood of the same patients. In contrast, however, in the synovial fluid the V delta 1 and the V delta 2 subsets differed with respect to the expression of the two isoforms of the leucocyte common antigen. The majority of the V delta 1+ cells expressed the high molecular weight isoform (CD45RA+) while most of the V delta 2+ cells carried the low molecular weight variant (CD45RO+) of this molecule.

Immunosuppression by seminal prostaglandins.

In this paper we report studies undertaken to determine the contribution of seminal prostaglandins to some of the known immunosuppressive properties of human seminal plasma. Initial studies revealed that fractions of seminal plasma enriched in E series prostaglandins, obtained by reverse phase chromatography, had a pronounced inhibitory effect on the PHA-induced proliferation of peripheral blood lymphocytes and on the NK-cell-mediated lysis of K562 target cells. Additional investigations revealed that similar inhibitory effects could be achieved with purified PGE2 (10(-6) to 10(-9) M) and 19-OH PGE1 (10(-6) to 10(-7) M), both of which are present in uniquely high concentrations in human seminal plasma. In contrast, 19-OH PGF1 which is found in lower concentrations in semen was slightly stimulatory in proliferative assays and had no effect on NK-cell-mediated cytotoxicity. Removal of the seminal prostaglandins by absorption chromatography resulted in a dramatic decrease in immune suppressive activity. Further studies with fractions obtained by ion-exchange HPLC of desalted seminal plasma indicated that prostaglandins complexed with seminal proteins, and these too were immunosuppressive. The possible relevance of these results to sexually transmitted disease is discussed.

Optimized assay for antisperm cell-mediated immunity.

Previous studies on antisperm cell-mediated immunity (CMI) have been confounded by the presence of immunogenic leukocytes in sperm antigen preparations. In this study we isolated pure populations of viable spermatozoa on discontinuous Percoll gradients, and utilized sonicated and cavitated extracts, as well as live motile spermatozoa, to measure cellular immunity to spermatozoa in vasectomized men, men with proven fertility, infertile women, fertile women and umbilical cord blood. Using a thymidine incorporation assay to assess lymphocyte proliferation, nine out of 13 (69%) vasectomized men and five out of 10 (50%) fertile men responded to sperm extracts. Lymphocyte proliferation to sperm extracts was also observed in both infertile and fertile women (27 and 50% respectively). In addition, viable sperm preparations promoted lymphocyte responses in five out of eight (63%) fertile women, seven out of 11 (63%) healthy men and four out of 11 (45%) cord blood specimens. Furthermore, four out of 11 (36%) healthy normal men responded to autologous spermatozoa. No relationship between serum antisperm antibodies, as measured with the Immunobead test, and sperm CMI was observed in any group. This study provides evidence that lymphocytes from fertile as well as infertile men and women and sperm-naive newborn infants proliferate when exposed to viable spermatozoa or sperm extracts. Thus the lymphocyte proliferation assay does not appear to be useful in the diagnosis of immunological infertility, but immunological recognition of spermatozoa may be a common feature that could have a role in fertility.

T lymphocytes and macrophages, but not motile spermatozoa, are a significant source of human immunodeficiency virus in semen.

The cellular fraction of semen contains spermatozoa, immature germ cells, leukocytes, and epithelial cells. Recent evidence implicates seminal cells as a major source of sexually transmitted human immunodeficiency virus (HIV) in semen, but the identity and infectious potential of infected cells remains poorly understood. HIV provirus was found in 75% of viable semen cell samples by polymerase chain reaction and in 88% of paired blood cell samples from HIV-seropositive men. When semen cell subpopulations were isolated by an immunomagnetic bead technique, T cells were found to be most commonly HIV-infected (75% of samples), followed by macrophages (38% of samples). Viral DNA was never detected in motile spermatozoa or immature germ cell populations. Semen leukocytes proliferated in response to mitogenic and antigenic challenge and produced p24 following stimulation with irradiated allogeneic cells. These data provide evidence that both T cells and macrophages, but not germ cells, are cellular vectors of HIV transmission in semen.

Superantigen-mediated proliferation and cytotoxicity of T cells isolated from the inflammatory tissues and peripheral blood of arthritis patients.

Superantigens are thought to play a role in acute infections and in the pathogenesis of autoimmune diseases that are believed to have an infectious etiology. The effect of the superantigens staphylococcal enterotoxin A, staphylococcal enterotoxin B, and streptococcal M type 5 protein on T cells derived from inflammatory tissues and peripheral blood (PB) of arthritis patients was studied in seven rheumatoid arthritis (RA), two psoriatic arthritis, two reactive arthritis, and one ankylosing spondylitis patient. Superantigen-reactive T cells and T cell lines derived from the PB, synovial fluid (SF), and synovial membrane (SM) of all 12 arthritis tissues recognized the superantigens in an MHC-unrestricted manner. Heterogeneities in proliferation and superantigen-directed T cell cytotoxicity were observed in E+ T cells and the T cell lines. Four SF-CD4+ mycobacteria heat-shock protein 65-kDa specific T cell clones generated from an RA patient could recognize and lyse each other when pulsed with staphylococcal enterotoxin A and used as targets. From another RA patient, four SF-CD4+ T cell clones that specifically recognize autoantigens were generated with human IgG fragments or collagen type II fragments. Heterogeneities of such superantigen-mediated specific lysis were also demonstrated. The data presented by us suggest a model in which superantigens do not have to be involved in triggering the initial disease because autoreactive T cells elicited by antigen can, in the presence of superantigen, lyse cells that express MHC class II molecules, including activated T cells.

Rheumatoid inflammatory T-cell clones express mostly Th1 but also Th2 and mixed (Th0-like) cytokine patterns.

This study was performed in order to characterize whether T cells from rheumatoid synovial inflammation belong to the Th1- or Th2-like functional subsets. Cytokine production was studied in 26 CD4+ alpha beta+ and 2 CD8+ alpha beta T-cell clones from the synovial fluid, the synovial membrane and peripheral blood of 5 patients. Fifteen of the CD4+ clones were raised against various mycobacterial antigens and 11 CD4+ clones and 2 CD8+ clones were raised unspecifically using PHA and/or IL-2. The specificities of these clones are not known. In the mycobacterial antigen-specific group, all CD4+ alpha beta T-cell clones produced IFN-gamma at high levels, while the production of IL-4 was generally absent or low (< 1 ng/ml), consistent with a Th1-like profile. Some of these clones, however, also produced various amounts of IL-10 which has been regarded as a Th2 product but can be produced also in lower amounts by Th1 cells. One HSP-65-specific clone produced levels of IL-4 and IL-10 in the same order as that of IFN-gamma, thus appearing to be Th0-like. Among the 11 unspecific CD4+ clones, 7 showed a Th1-like pattern but with lower levels of IFN-gamma than the antigen-specific clones. However, three clones did not produce any IFN-gamma activity but produced IL-4 and one of them also produced distinct amounts of IL-10, compatible with a Th2-like pattern. In addition, one of the clones also showed an almost equally strong IFN-gamma and IL-4 production, thus most likely representing a Th0-like clone.