TLR2介导沙眼衣原体感染早期免疫应答的机制研究

目的 探讨TLR2在小鼠沙眼衣原体生殖道早期感染时的免疫应答中的作用.方法 沙眼衣原体小鼠肺炎株(MoPn)经生殖道分别感染野生型小鼠(WT=11只)、TLR2基因缺陷小鼠(TLR2 KO=14只),建立生殖道沙眼衣原体感染模型.分别于感染后不同时间点取阴道棉拭子,获取分泌物,检测分泌物衣原体包涵体数量和炎症细胞因子IL-1α、IL-6和MIP-2水平.结果 TLR2 KO和WT小鼠在每个检测时间点,生殖道分泌物带菌量无差异,且带菌持续时间相同;与WT小鼠比较,在感染后第3d和第10d,TLR2 KO小鼠生殖道分泌物的炎症细胞因子IL-1α、IL-6和MIP-2水平均低于野生型WT小鼠,但差异均无统计学意义(P＞0.05);而在感染后第7d,3种细胞因子均明显低于WT小鼠,且差异有统计学意义(P＜0.05).结论 在沙眼衣原体生殖道感染中,TLR2可能介导了早期炎症细胞因子的产生.     

Immune Regulation by T Cell Subsets and their Cytokines

Inflammation Research -     

On the Molecular Basis of T Helper Cell Function

B-lymphocyte promotor factors (B-LPF) are defined as T-cell-derived, released molecules that trigger polyclonal induction of B-cell differentiation into antibody-forming cells. B-LPF activity is independent of antigen, and it apparently induces only IgM-producing B cells. B-LPF was discovered as products of an antigen-specific, I-Ab-restricted T-cell line. We here show that B-LPF is produced also by lymphoma cells derived from this T-cell line or by T-cell hybrids constructed by fusing the T-cell line with BW5147 thymoma cells. A chicken gamma globulin-specific T-cell hybridoma clone also produced B-LPF. Biological assays demonstrated that B-LPF-containing supernatants did not contain IL-1, IL-2, B-cell growth factor, or allogeneic effector factor. Biochemical studies showed that B-LPF was precipitated by 50% (NH4)2SO4 saturation and that at least three types of molecules were involved in B-LPF activity: molecules with molecular weights of greater than 90,000, 50,000-90,000 and 10,000-25,000. The relationship between B-LPF and antigen-specific helper/inducer factors is discussed.     

On the Molecular Basis of T Helper Cell Function

The differentiation of Ig⁺ B cells into plaque-forming cells is dependent on antigen and factors produced by T cells and/or macrophages. We describe here the production of T-cell factors termed lymphocyte promotor factors (LPF). A foetal calf serum-specific T-cell line and its clones synthesize LPF, which is defined as factors that polyclonally stimulate normal spenic T cells to differentiate into cytotoxic T lymphocytes (T-LPF) and normal splenic B cells to differentiate into plaque-forming cells into (PFC) (B-LPF) in the apparent absence of specific antigen. The proliferation of and the B-LPF production of all T-cell clones tested were foetal calf serum-specific and I-A^b-restricted. Some of these clones produced only T-LPF, some clones produced only B-LPF, and some clones produced both T-LPF and B-LPF. B-LPF stimulate the polyclonal differentiation of Ig⁺ B cells into PFC without the apparent need for helper T cells, is different from T-LPF, and induces almost exclusively IgM PFC. The B-LPF described in the present paper are compared with previously described T-cell factors, which stimulate antigen-specific B-cell responses or bystander B-cell responses. The conclusion is that B-LPF are probably different from B-cell growth factors, T-cell replacing factors, allogeneic effector factors, and interleukin 2.     

Chemokine Expression Patterns in the Systemic and Genital Tract Compartments are Associated with HIV-1 Infection in Women from Benin

Introduction  

Understanding the genital mucosal immunity and the factors involved in linking innate to adaptive immunity is crucial for the design of efficient preventive strategies against human immunodeficiency virus (HIV)-1.     

Mouse Strain-Dependent Differences in Susceptibility to Neisseria gonorrhoeae Infection and Induction of Innate Immune Responses

Acute gonorrhea in women is characterized by a mucopurulent exudate that contains polymorphonuclear leukocytes (PMNs) with intracellular gonococci. Asymptomatic infections are also common. Information on the innate response to Neisseria gonorrhoeae in women is limited to studies with cultured cells, isolated immune cells, and analyses of cervicovaginal fluids. 17β-Estradiol-treated BALB/c mice can be experimentally infected with N. gonorrhoeae, and a vaginal PMN influx occurs in 50 to 80% of mice. Here, we compared the colonization loads and proinflammatory responses of BALB/c, C57BL/6 and C3H/HeN mice to N. gonorrhoeae. BALB/c and C57BL/6 mice were colonized at similar levels following inoculation with 10⁶ CFU of N. gonorrhoeae. BALB/c, but not C57BL/6, mice exhibited a marked vaginal PMN influx. Tumor necrosis factor alpha, interleukin-6, macrophage inflammatory protein 2 (MIP-2), and keratinocyte-derived chemokine were elevated in vaginal secretions from infected BALB/c mice, but not in those from C57BL/6 mice. MIP-2 levels positively correlated with a vaginal PMN influx. In contrast to BALB/c and C57BL/6 mice, C3H/HeN mice were resistant to infection, and there was no evidence of an inflammatory response. We conclude that N. gonorrhoeae causes a productive infection in BALB/c mice that is characterized by the induction of proinflammatory cytokines and chemokines and the recruitment of PMNs. Infection of C57BL/6 mice, in contrast, is more similar to asymptomatic infection. C3H/HeN mice are inherently resistant to N. gonorrhoeae infection, and this resistance is not due to an overwhelming inflammatory response to infection. Host genetic factors can therefore impact susceptibility and the immune response to N. gonorrhoeae.Uncomplicated gonorrhea is most commonly an infection of the urethra in men and the cervix. The female urethra may also be infected, and rectal and pharyngeal infection can occur in either sex. The hallmark of symptomatic gonococcal infection is the presence of a purulent exudate containing numerous polymorphonuclear leukocytes (PMNs), many of which contain intracellular gonococci. Asymptomatic infections are also common, particularly in females. Epithelial cells that line the genital mucosal surface are the first line of defense against this human-specific pathogen, and Neisseria gonorrhoeae produces a robust proinflammatory cytokine and chemokine response when incubated with cultured human vaginal, endocervical, ectocervical (12, 33), urethral (17), endometrial (3), and fallopian tube (31) tissue culture cells. Similarly, studies using the complex fallopian tube organ culture model suggest that N. gonorrhoeae induces the proinflammatory cytokines interleukin-1α (IL-1α) and tumor necrosis factor alpha (TNF-α) (29). Signaling through cellular receptors on epithelial cells results in the activation and recruitment of phagocytic cells, including PMNs and macrophages. Primary macrophages and peripheral blood mononuclear cells also elicit a proinflammatory response when incubated with N. gonorrhoeae (30, 34, 39). These innate immune cells further contribute to the array of proinflammatory cytokines and antimicrobial factors.Due to the multiple cell types that contribute to the host innate response to infection, it is important that whole model systems be utilized to measure the impact of N. gonorrhoeae infection on the host immune response. Experimental urethral colonization in male volunteers with N. gonorrhoeae evokes a strong innate response that is characterized by the production of proinflammatory cytokines (37, 38). Similar studies with female subjects are not feasible due to the risk of complications of gonococcal infection in women. Therefore, features of the innate response to N. gonorrhoeae in the female genital tract are predicted solely from tissue culture systems and the analysis of clinical samples. It is unclear whether women elicit a cytokine response to gonococcal infection. The reason why some infections are asymptomatic is also not known. Hedges et al. (18, 19) were unable to detect local proinflammatory cytokines in cervicovaginal secretions from infected women and detected a low anti-gonococcal antibody response. Based on these observations, it was proposed that N. gonorrhoeae fails to induce host inflammatory responses or is actively immunosuppressive. This finding is in marked contrast with the robust induction of proinflammatory cytokines observed from in vitro cell lines that constitute the female genital tract. The absence of various other cell types in tissue culture cell models could influence the cytokine response to infection; alternatively, the timing of sample collection from infected subjects may also influence the data. Therefore, a systematic analysis of cytokine induction over the course of infection in a female animal model is needed.The 17β-estradiol-treated mouse model is the only small-animal model available for studying the immune response to N. gonorrhoeae genital tract infection. While the mechanism by which estradiol promotes long-term colonization in female mice is not known, it is likely that promotion of an estrus-like state is beneficial for the gonococcus based on the fact that untreated mice can be transiently colonized with N. gonorrhoeae provided they are inoculated in the proestrus stage of the reproductive cycle (7, 46). The 17β-estradiol-treated mouse model has been a useful system for studying many aspects of gonococcal infection, including gonococcal evasion of PMN killing (43, 49) and antimicrobial peptides (23, 48), antigenic variation in vivo (41), and interactions between N. gonorrhoeae and commensal flora (32). This model is based on the use of BALB/c mice. Approximately 50 to 80% of infected BALB/c mice that are treated with a slow-release estradiol pellet exhibit a significant vaginal PMN response following inoculation with N. gonorrhoeae strain FA1090 based on examination of stained vaginal smears (21, 22, 43), and PMNs and macrophages are also found in vaginal and cervical tissue samples from infected mice (44). Gonococci are localized within vaginal and cervical tissue, and similar to that which occurs in humans, an insignificant and transient humoral response to N. gonorrhoeae occurs which was not protective against reinfection with the same strain (44). A recent modification of the model utilizes water-soluble estradiol to reduce the length of time that mice are exposed to nonphysiological concentrations of estradiol. A vaginal PMN influx also occurs during infection of mice treated with water-soluble estradiol, and as with pelleted mice, infection persists despite the presence of PMNs (44).One advantage of using inbred mouse strains for studies of infectious diseases is that environmental and genetic components can be controlled. Interestingly, the susceptibility to infectious agents can often vary with the genetic background of the mouse. One example in the area of sexually transmitted infections is that genetically controlled differences in the development of infertility in inbred mouse strains following inoculation with chlamydia have been reported, with pregnancy rates following infection of C3H/HeN mice being significantly lower than those of C57BL/6 mice (10). Darville and colleagues (8) found similar results, and their data suggested that an earlier and more severe acute inflammatory response in the C57BL/6 strain may lead to earlier eradication of the infection, thus protecting the upper tract from disease. Numerous examples of vulnerability have been found for other infectious agents, including Leishmania major, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Plasmodium chabaudi, Legionella pneumophila, and Mycobacterium tuberculosis (reviewed by Kramnik and Boyartchuk [27]).In this study, we sought to characterize in greater detail the cytokine and inflammatory response to genital tract infection with N. gonorrhoeae in 17β-estradiol-treated BALB/c mice and to determine if susceptibility to colonization and the host inflammatory response to infection vary between inbred mouse strains. Our data demonstrate that BALB/c, C57BL/6, and C3H/HeN mouse strains differ widely in their response to infection. While both BALB/c and C57BL/6 strains support colonization with the gonococcus, only the BALB/c strain appears to mount an inflammatory response. In contrast, the C3H/HeN strain appears to be resistant to colonization with the gonococcus. These data demonstrate significant divergence among inbred mouse strains in terms of susceptibility and inflammatory response to gonococcal infection, and they suggest that future studies can be designed to correlate genetic markers with the host response.     

T Helper Cell Control of B Cell Development and Isotype Expression

The supernatant of the T helper cell clone 52.3 (52.3-SN) was shown to induce polyclonal activation of resting B cells. 52.3-SN acts on most small B cells and through the allogeneic barrier. This supernatant induces cell size increase, RNA and DNA synthesis, and appearance of interleukin-2 and transferrin receptor. These results are interpreted as indicating the existence of a B Cell Activating Factor (BCAF) acting on resting B cells in an MHC-unrestricted way.

TH cells can be obtained in an intermediate state of activation where they secrete lymphokines leading to B cell proliferation and not the biological activities leading to plasmocyte development. TH cell clones can induce sIgG^? and sIgA^? unprimed B cells to switch and express all classes and subclasses of immunoglobulin. The bulk of the response consists of IgM. Among the non IgM isotypes, IgG₁ and IgA predominate. The supernatants prepared from TH cells reproduce these effects.     

Type 1 Fimbrial Adhesin FimH Elicits an Immune Response That Enhances Cell Adhesion of Escherichia coli

Escherichia coli causes about 90% of urinary tract infections (UTI), and more than 95% of all UTI-causing E. coli express type 1 fimbriae. The fimbrial tip-positioned adhesive protein FimH utilizes a shear force-enhanced, so-called catch-bond mechanism of interaction with its receptor, mannose, where the lectin domain of FimH shifts from a low- to a high-affinity conformation upon separation from the anchoring pilin domain. Here, we show that immunization with the lectin domain induces antibodies that exclusively or predominantly recognize only the high-affinity conformation. In the lectin domain, we identified four high-affinity-specific epitopes, all positioned away from the mannose-binding pocket, which are recognized by 20 separate clones of monoclonal antibody. None of the monoclonal or polyclonal antibodies against the lectin domain inhibited the adhesive function. On the contrary, the antibodies enhanced FimH-mediated binding to mannosylated ligands and increased by severalfold bacterial adhesion to urothelial cells. Furthermore, by natural conversion from the high- to the low-affinity state, FimH adhesin was able to shed the antibodies bound to it. When whole fimbriae were used, the antifimbrial immune serum that contained a significant amount of antibodies against the lectin domain of FimH was also able to enhance FimH-mediated binding. Thus, bacterial adhesins (or other surface antigens) with the ability to switch between alternative conformations have the potential to induce a conformation-specific immune response that has a function-enhancing rather than -inhibiting impact on the protein. These observations have implications for the development of adhesin-specific vaccines and may serve as a paradigm for antibody-mediated enhancement of pathogen binding.     

Heterogeneity in the Specificities of an Alloreactive T Helper Cell Line

Heterogeneity in antigen recognition of an alloreactive helper T-cell line was studied by repeated limiting dilution cloning. Analysis of the fine specificities of these clones showed that the gain of reactivity to new antigens by the T-cell line included the generation of non-cross-reactive T cells specific to new major histocompatibility (MHC) antigens. Analysis of function revealed that all the T-cell clones studied had similar cell surface antigens and secreted interleukin 4 (IL-4) and IL-5/IL-3 but not IL-2 or gamma interferon (IFN-gamma). The T-cell clones served as helper cells for B cells expressing the appropriate MHC antigens to both clonal proliferation and differentiation into antibody-producing cells.