DA rat NK(+)CD3(-) cells inhibit autoreactive T-cell responses.

We isolated natural killer (NK) cells from DA rat bone marrow (BM) by staining with PE anti-NKR-P1A and FACS sorting (>98% NK(+)). The purified NK cells inhibit T-cell proliferation in a dosage-dependent fashion and suppressed production of the proinflammatory Th1 cytokine, IFN-gamma. When activated in vitro with Con A supernatant (CAS), the purified NK cells secrete the beta-chemokine monocyte chemoattractant protein-1 (MCP-1) and upregulate MCP-1 mRNA. The activated NK cells also express IFN-gamma mRNA. Sorted NK(+)CD3(-) cells, from which NKT cells have been excluded, also inhibit autoreactive T-cell responses to myelin basic protein (MBP). These findings are consistent with a role for conventional NK cells in maintaining immune homeostasis, by eliminating autoreactive T cells that have inadvertently become activated.

A Chlamydia pneumoniae-specific peptide induces experimental autoimmune encephalomyelitis in rats.

It has been reported recently that the bacterial respiratory pathogen Chlamydia pneumoniae is present in the cerebrospinal fluid of a subset of multiple sclerosis (MS) patients. However, it is not known whether this organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. We report identification of a 20-mer peptide from a protein specific to C. pneumoniae which shares a 7-aa motif with a critical epitope of myelin basic protein, a major CNS Ag targeted by the autoimmune response in MS. This bacterial peptide induces a Th1 response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective of many aspects of MS. Studies with peptide analogues suggest that different populations of encephalitogenic T cells are activated by the C. pneumoniae and myelin basic protein Ags. Mild experimental autoimmune encephalomyelitis was also observed when rats were immunized with sonicated C. pneumoniae in CFA.

Parental genes do not codominantly confer susceptibility to experimental autoimmune encephalomyelitis in F1 rats.

Lewis (LEW) and DA rats are highly susceptible to experimental autoimmune encephalomyelitis (EAE) induced with guinea pig myelin basic protein (MBP), but respond to different epitopes. The dominant epitope for LEW rats is MBP73-86, and disease is mediated primarily by Vbeta8.2 Th1 cells. DA rats lack conventional Vbeta8.2 T cells and do not respond to MBP73-86. Rather, DA rats respond to the cryptic epitope MBP63-81, which is not encephalitogenic for LEW rats. Responses to these neuroantigens were investigated in (DAxLEW) F1 hybrids to determine if experimental findings in inbred rats remain valid in more genetically complex models. Surprisingly, MBP63-81, a cryptic epitope for DA rats, induced moderate-to-severe EAE in F1 hosts, whereas MBP73-86, the dominant LEW epitope, was only weakly encephalitogenic in F1 hosts. The poor clinical response to MBP73-86 appears to be a consequence of an inability to expand Vbeta8.2 T cells. These results suggest that parental responses to neuroantigens are poor predictors for determining encephalitogenicity in F1 progeny.

Candida-specific systemic cell-mediated immune reactivities in human immunodeficiency virus-positive persons with mucosal candidiasis.

Oropharyngeal candidiasis (OPC), as opposed to vulvovaginal candidiasis (VVC), is a common opportunistic infection in human immunodeficiency virus (HIV)-positive persons that correlates with reduced CD4 T cell counts. Although cell-mediated immunity (CMI) by CD4 Th1-type cells is considered to be the predominant host defense against mucosal candidiasis, the immune factors associated with susceptibility to OPC in HIV-positive persons are not well understood. This study investigated Candida-specific systemic CMI in HIV-positive persons with OPC and/or VVC. Reductions in delayed skin test reactivity to Candida antigen were observed in HIV-positive persons with CD4 cell counts <200 cells/microL, irrespective of the presence of mucosal infection. Likewise, despite the correlate of OPC with reduced CD4 cell counts in HIV-positive persons, differences in Candida-specific peripheral blood mononuclear cell proliferation and Th1/Th2 cytokine production between HIV-positive and HIV-negative persons were not consistent in a manner to suggest that deficiencies in Candida-specific systemic CMI account solely for the susceptibility to OPC.

Strain variation in autoimmunity: attempted tolerization of DA rats results in the induction of experimental autoimmune encephalomyelitis.

This paper reports that DA rats develop experimental autoimmune encephalomyelitis (EAE) when immunized with encephalitogenic myelin basic protein (MBP) peptide (MBP63-81) in IFA. In contrast, most rodent strains are tolerized by this procedure. Doses as low as 5 micrograms peptide + IFA induced EAE in DA rats. Lewis (LEW) rats did not develop EAE, even after immunization with 100 micrograms encephalitogenic peptide (MBP68-86) + IFA, but were rendered tolerant to EAE. DA rat T cells proliferated to peptide, and proliferation was inhibited by CTLA4Ig, and by anti-B7.1 and anti-B7. 2 mAbs. This indicates that the ease of induction of EAE in this strain does not reflect a decreased requirement for T cell costimulation through the B7/CD28 costimulatory pathway. The inhibitory effect of CTLA4Ig was abrogated in the presence of anti-TGF-beta-neutralizing Ab. An encephalitogenic DA T cell line expressed mRNA for the Th1 cytokines IFN-gamma and TNF-alpha, as well as IL-10, and secreted these cytokines. In contrast, a T cell line from peptide + IFA-immunized LEW rats (which did not develop EAE) failed to secrete these cytokines. Although this line did not express TNF-alpha or IL-10 mRNA, IFN-gamma mRNA was detected, suggesting posttranscriptional regulation of IFN-gamma expression. Attempts to induce unresponsiveness in DA rats with encephalitogenic peptide-coupled splenocytes were also unsuccessful.

Inhibition of autoimmune T cell responses in the DA rat by bone marrow-derived NK cells in vitro: implications for autoimmunity.

Regulation of the immune response is critical to homeostasis. While innate immunity can influence the development of adaptive immune responses, its role in regulation is less well understood. Recently, NK cells have been implicated in the control of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. In this report, we show that rat bone marrow-derived NK cells exhibited potent inhibitory effects on T cell proliferation to both Con A as well as the central nervous system Ag myelin basic protein. There was also a significant decrease in both IFN-gamma and IL-10 production in vitro, whereas levels of the beta-chemokine monocyte chemoattractant protein-1 were significantly elevated. Flow cytometry studies suggest that the NK cells may play an important role in regulating both normal and autoimmune T cell responses by exerting a direct effect on activated, autoantigen-specific T cells.

Critical requirement for aspartic acid at position 82 of myelin basic protein 73-86 for recruitment of V beta 8.2+ T cells and encephalitogenicity in the Lewis rat.

We synthesized single amino acid-substituted peptide analogues of guinea pig myelin basic protein (MBP) 73-86 to study the importance of aspartic acid at residue 82 (QKSQRSQDENPV), which previous reports have suggested is a critical TCR contact residue. Whereas the wild-type 73-86 peptide elicited severe experimental autoimmune encephalomyelitis (EAE) in the Lewis rat, none of the peptide analogues with substitutions at position 82 were capable of inducing EAE. The inability to cause EAE was not due to a failure to bind MHC or to elicit T cell proliferation and cytokine secretion. T cells specific for MBP73-86 did not cross-react with any of the analogues tested, further indicating the importance of this residue in T cell responses to 73-86. Analysis by flow cytometry showed that only the wild-type 73-86 peptide was capable of recruiting V beta 8.2+ T cells, which have been shown previously to be important for disease induction. Reduced expression of the V beta 8.2 TCR was also seen in Lewis rats protected from EAE by coimmunization of MBP73-86 with 73-86(82D-->A), despite an increase in cytokine production when both peptides were present during in vitro culture. The data indicate that aspartic acid 82 is a critical TCR contact residue and is required for the recruitment of V beta 8.2+ T cells and the encephalitogenic activity of MBP73-86.

Delineation of two encephalitogenic myelin basic protein epitopes for DA rats.

We studied synthetic peptides that correspond to two regions of the guinea pig myelin basic protein (MBP) molecule which elicit experimental autoimmune encephalomyelitis (EAE) in DA rats. Using truncated peptides, we determined that two encephalitogenic epitopes reside within MBP63-81, a major determinant defined by MBP residues, 63-76, and a minor encephalitogenic epitope defined by residues, 66-81. Experiments with alanine-substituted analogs of MBP63-76 revealed that the HYGSLP sequence is critical for encephalitogenicity. The core epitope within a second encephalitogenic region, MBP101-120, was defined by residues, 106-119. Studies with analogs of this sequence indicated that residues, Leu 111, Phe 114 and Trp 116 are important for T-cell responsiveness.

Interstrain variability of autoimmune encephalomyelitis in rats: multiple encephalitogenic myelin basic protein epitopes for DA rats.

We investigated T cell epitopes of guinea pig myelin basic protein (MBP) that induce experimental autoimmune encephalomyelitis (EAE) in DA rats, using synthetic peptides that correspond to regions of the guinea pig MBP molecule that are homologous to rat MBP. Four peptides were encephalitogenic when tested in DA rats. MBP63-81, which partially overlaps the dominant encephalitogenic MBP epitope for Lewis (LEW) rats, caused severe EAE in the DA strain but did not elicit EAE in LEW rats. MBP66-81 and MBP63-76 were also encephalitogenic for DA but not LEW rats. MBP79-99 also induced EAE in DA rats, although MBP87-99, the minor encephalitogenic LEW epitope, was inactive. This indicates that part of the 79-86 sequence is necessary for encephalitogenic activity in the DA strain. MBP101-120, and MBP142-167 were also encephalitogenic for DA rats. T cells from DA rats immunized with intact MBP proliferated in response to the whole protein and to MBP79-99, but were not stimulated to a significant extent by the other encephalitogenic peptides, suggesting that these may represent cryptic or subdominant epitopes. However, MBP63-81-specific T cell lines could be isolated by repeated restimulation with peptide, indicating that the peptide-specific T cells were present in DA rats at low frequency.

Use of cellular depletion analysis to examine circulation of immune effector function between the vagina and the periphery.

Results from an animal model of vaginal candidiasis suggest that Candida-specific cell-mediated immunity in the systemic circulation does not mediate protection against vaginitis. The present study used cellular depletion analysis to examine the circulation of immune effector function between the vagina and the periphery. Results showed that anti-Thy-1.2 antibodies given intravenously to mice depleted Thy-1+ T lymphocytes in the systemic compartment but not in the vaginal mucosa, while the same antibodies injected intravaginally significantly reduced Thy-1+ T cells in both the vaginal and systemic compartments. These results support a lack or low level of circulation of immune effector function from the periphery to the vaginal mucosa.

Vaginal-associated immunity in women with recurrent vulvovaginal candidiasis: evidence for vaginal Th1-type responses following intravaginal challenge with Candida antigen.

Studies from women with recurrent vulvovaginal candidiasis (RVVC) and from an animal model of experimental vaginitis suggest that deficiencies in immune function should be examined at the local rather than systemic level. Evidence of vaginal cell-mediated immunity (CMI) was evaluated for the first time in cervicovaginal lavage (CVL) fluid from RVVC patients. Results showed that although constitutive Th1- and Th2-type cytokine expression was detectable in CVL fluid from normal women, and differences in cytokines were observed in RVVC patients, limitations in experimental design of such de novo analyses urged caution in interpretation. Alternatively, attempts were made to establish conditions in control subjects whereby vaginal immunity could be detected after intravaginal challenge with Candida antigen. Preliminary results showed that Th1-type cytokines (interleukin-2 and -12, interferon-gamma) and histamine were increased 16-18 h after intravaginal introduction of Candida skin test antigen. Intravaginal antigenic challenge represents a novel approach for studying Candida-specific vaginal CMI.

Detection of human defensin 5 in reproductive tissues.

OBJECTIVES: This study was undertaken to determine whether tissue-specific defensins are expressed within female reproductive tissues. STUDY DESIGN: Messenger ribonucleic acid from amnion, chorion, endometrium, endocervix, myometrium, placenta, small intestine, peripheral blood lymphocytes, and cervical, endometrial, and trophoblast cell lines was reverse transcribed with a 3'-RACE adapter. 3'-RACE polymerase chain reaction was conducted with an upstream human defensin 5 primer and 3'-RACE adapter primer. Polymerase chain reaction products hybridizing to a human defensin 5 probe were cloned for sequence analysis. Sequence data were compared against a nucleotide sequence database, and secondary structure predictions were made. RESULTS: Chorionic tissue, endocervical tissue, endometrial tissue, and an endometrial cell line all demonstrated a single hybridizing 362 bp polymerase chain reaction product. Sequence analysis of all clones demonstrated near-perfect identity with human defensin 5. CONCLUSIONS: Human endocervix, endometrium, and chorion express defensin 5 at the level of transcription. These findings suggest that a previously unrecognized mechanism of protecting female reproductive tissues against infection, by means of a natural antimicrobial system (defensins), may be present.

Potential effects of midcycle cervical mucus on mediators of immune reactivity.

OBJECTIVE: To examine the effect of cervical mucus (CM) on immune mediators in cervicovaginal lavage fluid. DESIGN: The stability of cytokines and immunoglobulins (Ig) within cervicovaginal lavage fluid was determined during 18-hour contact with unsolubilized midcycle CM in vitro. SETTING: In vitro experiment. PATIENT(S): Healthy female volunteers from whom cervicovaginal lavage fluid and periovulatory CM were obtained. INTERVENTION(S): Changes in cytokine and Ig concentration in cervicovaginal lavage fluid after incubation with CM. MAIN OUTCOME MEASURE(S): Interleukin (IL)-4, IgA, and IgG concentrations were determined by ELISA. Interleukin-2 concentration was determined by bioassay using the IL-2-dependent cytotoxic T-lymphocyte cell line and ELISA. Results were expressed as percent antibody or cytokine recovery remaining after mucus contact, with differences over time evaluated by analysis of variance. RESULT(S): During 18-hour mucus contact, significant changes were detected for IL-4, IgA, and IgG concentration. Interleukin-2 was stable in mucus-associated cervicovaginal lavage fluid as determined by functional bioassay and ELISA. CONCLUSION(S): Immunoglobulins and cytokines differ in their recovery from aqueous media after CM contact, suggesting that midcycle CM could alter immune reactivity within the reproductive tract by modifying the availability or function of immunomodulatory substances.

T lymphocytes in the murine vaginal mucosa are phenotypically distinct from those in the periphery.

The results from both clinical studies of women with recurrent vulvovaginal candidiasis and a murine model of experimental vaginitis indicate that systemic cell-mediated immunity may not represent a dominant host defense mechanism against vaginal infections by Candida albicans. Recent experimental evidence indicates the presence of local vaginal immune reactivity against C. albicans. The present study was designed to examine T-lymphocyte subpopulations in the vaginal mucosae of naive CBA/J mice. Vaginal lymphocytes (VL) were isolated by collagenase digestion of whole vaginal tissues. Cell populations were identified by flow cytometry, and the results were compared with those for both lymph node cells (LNC) and peripheral blood lymphocytes (PBL). The results of flow cytometry showed that 45% +/- 10% of lymphocytes in the vaginal mucosa are CD3+ compared with 75% +/- 5% in LNC and 50% +/- 5% in PBL. The majority (85%) of CD3+ VL are CD4+ and express the alpha/beta T-cell receptor (TCR), similar to the results for LNC and PBL. In contrast to LNC and PBL, VL contain a significantly higher percentage (15 to 20%) of gamma/delta TCR+ cells, 80% or more of which appear to express CD4. In addition, while CD4-CD8 cell ratios in LNC and PBL were 3:1 and 6:1, respectively, only 1% of VL expressed CD8, resulting in a CD4-CD8 cell ratio of > 100:1. Finally, while LNC and PBL recognized two epitope-distinct (GK 1.5 and 2B6) anti-CD4 antibodies, VL recognized only 2B6 anti-CD4 antibodies. Further analysis of VL showed that Thy-1 cells, but not CD4 cells, were reduced after intravaginal injection of complement-fixing anti-Thy-1.2 and GK 1.5 anti-CD4 antibodies, respectively. Taken together, these data suggest that T lymphocytes in the vaginal mucosae of mice are phenotypically distinct from those in the periphery and that CD4+ VL have an uncharacteristic or atypical expression of the CD4 receptor.