Global gene expression of a murein (Braun) lipoprotein mutant of Salmonella enterica serovar Typhimurium by microarray analysis.

Braun/murein lipoprotein (Lpp) is one of the major outer membrane components of gram-negative enteric bacteria involved in inflammatory responses and septic shock. In previous studies, we reported that two copies of the lipoprotein (lpp) gene (designated as lppA and lppB) existed on the chromosome of Salmonella enterica serovar Typhimurium. Deletion of both lppA and lppB genes rendered Salmonella defective in invasion, motility, induction of cytotoxicity, and production of inflammatory cytokines/chemokines. The lppAB double-knockout (DKO) mutant was attenuated in mice, and animals immunized with this mutant were protected against subsequent challenge with lethal doses of wild-type (wt) S. Typhimurium. To better understand how deletion of the lpp gene might affect Salmonella virulence, we performed global transcriptional profiling of the genes in the wt and the lppAB DKO mutant of S. Typhimurium using microarrays. Our data revealed alterations in the expression of flagellar genes, invasion-associated type III secretion system genes, and transcriptional virulence gene regulators in the lppAB DKO mutant compared to wt S. Typhimurium. These data correlated with the lppAB DKO mutant phenotype and provided possible mechanism(s) of Lpp-associated attenuation in S. Typhimurium. Although these studies were performed in in vitro grown bacteria, our future research will be targeted at global transcriptional profiling of the genes in in vivo grown wt S. Typhimurium and its Lpp mutant.

Attenuation of Salmonella enterica Serovar Typhimurium by altering biological functions of murein lipoprotein and lipopolysaccharide.

We constructed Salmonella enterica serovar Typhimurium double-knockout mutants in which either the lipoprotein A (lppA) or the lipoprotein B (lppB) gene was deleted from an msbB-negative background strain by marker exchange mutagenesis. These mutants were highly attenuated when tested with in vitro and in vivo models of Salmonella pathogenesis.

Murein lipoprotein is a critical outer membrane component involved in Salmonella enterica serovar typhimurium systemic infection.

Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-alpha and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.

The two murein lipoproteins of Salmonella enterica serovar Typhimurium contribute to the virulence of the organism.

Septic shock due to Salmonella and other gram-negative enteric pathogens is a leading cause of death worldwide. The role of lipopolysaccharide in sepsis is well studied; however, the contribution of other bacterial outer membrane components, such as Braun (murein) lipoprotein (Lpp), is not well defined. The genome of Salmonella enterica serovar Typhimurium harbors two copies of the lipoprotein (lpp) gene. We constructed a serovar Typhimurium strain with deletions in both copies of the lpp gene (lpp1 and lpp2) by marker exchange mutagenesis. The integrity of the cell membrane and the secretion of the effector proteins through the type III secretion system were not affected in the lpp double-knockout mutant. Subsequently, the virulence potential of this mutant was examined in a cell culture system using T84 intestinal epithelial and RAW264.7 macrophage cell lines and a mouse model of salmonellosis. The lpp double-knockout mutant was defective in invading and inducing cytotoxic effects in T84 and RAW264.7 cells, although binding of the mutant to the host cell was not affected when compared to the wild-type (WT) serovar Typhimurium. The motility of the mutant was impaired, despite the finding that the number of flagella was similar in the lpp double knockout mutant and the WT serovar Typhimurium. Deletion in the lpp genes did not affect the intracellular survival and replication of Salmonella in macrophages and T84 cells. Induction of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-8 (IL-8) was significantly reduced in macrophages and T84 cells infected with the lpp double-knockout mutant. The levels of IL-8 remained unaffected in T84 cells when infected with either live or heat-killed WT and lpp mutant, indicating that invasion was not required for IL-8 production and that Toll-like receptor 2 signaling might be affected in the Lpp double-knockout mutant. These effects of the Lpp protein could be restored by complementation of the isogenic mutant. The lpp double-knockout mutant was avirulent in mice, and animals infected with this mutant were protected from a lethal challenge dose of WT serovar Typhimurium. The severe combined immunodeficient mice, on the other hand, were susceptible to infection by the lpp double-knockout mutant. The serovar Typhimurium mutants from which only one of the lpp (lpp1 or lpp2) genes was deleted were also avirulent in mice. Taken together, our data indicated that Lpp specifically contributed to the virulence of the organism.

Protective immune response to hepatitis C virus in chimpanzees rechallenged following clearance of primary infection.

Hepatitis C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 years, suggesting that long-lasting protective immunity may be possible. The ability of peripheral blood mononuclear cells (PBMC) to recognize HCV proteins was evaluated during the course of the rechallenge experiments. A very early and strong in vitro recall response to HCV nonstructural proteins appeared to be associated with viral clearance. In contrast, proliferative responses to HCV proteins were not observed in 4 persistently infected chimpanzees, and a weak proliferative response was observed in 1 of 2 animals during acute resolving infection. The results suggest that a strong T-cell proliferative response is induced upon rechallenge of chimpanzees with HCV and that this response is associated with rapid viral clearance. The antibody response to HCV proteins increased by over 1,000-fold in all animals following rechallenge as well. A more complete understanding of the role of the cellular immune response in the clearance of HCV and the nature of the protective immune response following viral clearance may aid in the generation of therapies and vaccines.

Characterization of liver T-cell receptor gammadelta T cells obtained from individuals chronically infected with hepatitis C virus (HCV): evidence for these T cells playing a role in the liver pathology associated with HCV infections.

The pathogenic mechanisms involved in viral hepatitis are not completely understood. Evidence suggests that the pathology associated with hepatitis C virus (HCV) and hepatitis B virus (HBV) infections are a result of the immune response in the liver to these viruses. The livers of patients with viral hepatitis have been shown to contain elevated numbers of T cells expressing the gamma/delta form of the T-cell receptor for antigen (TCRgammadelta). In this study, we investigated whether liver biopsy specimens obtained from individuals with viral (HCV and/or HBV) or nonviral hepatitis contained TCRgammadelta(+) T cells that could be expanded in vitro by cytokines. A high percentage of liver biopsy specimens obtained from HCV- and/or HBV-infected individuals contained high numbers of TCRgammadelta(+) T cells. In contrast, T-cell lines generated from liver biopsy tissues obtained from individuals with nonviral hepatitis or from normal controls had no preferential expansion of TCRgammadelta(+) T cells. Liver TCRgammadelta(+) T-cell lines from HCV-infected individuals had high levels of non-major histocompatibility complex (MHC)-restricted cytotoxic activity against different targets including primary hepatocytes and produced interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin 8 (IL-8) following activation by anti-CD3. Surprisingly, none of these liver TCRgammadelta(+) T-cell lines could recognize any of the structural or nonstructural proteins of HCV and had no cytotoxic activity against cells infected with recombinant vaccinia viruses expressing different HCV proteins. However, the crosslinking of CD81, which has been shown to bind HCV particles and E2, resulted in significant levels of IFN-gamma and TNF-alpha production by liver TCRgammadelta(+) T cells. These results suggest that TCRgammadelta(+) T cells may play a role in the liver pathology of HCV infections.

Crosslinking CD81 results in activation of TCRgammadelta T cells.

CD81 is expressed on most cells and is associated with other glycoproteins, including CD4 and CD8, to form multimolecular membrane complexes. Crosslinking of CD81 on TCRalphabeta(+) T cells results in costimulatory signals that have been proposed to be mediated via CD4 or CD8. In this study, we show that CD81 is also expressed on TCRgammadelta(+)CD4(-)CD8(-) T cells. CD81 crosslinking greatly enhanced anti-CD3 activation of both TCRalphabeta(+) (CD4+ and CD8+) and TCRgammadelta(+) T cells with regard to IFN-gamma production. However, crosslinking of CD81 molecules on TCRgammadelta(+) T cells, in the absence of anti-CD3 stimulation, resulted in cytokine production and enhanced IL-2-induced proliferation, demonstrating that physical association with CD4 or CD8 is not necessary for CD81 signaling. In contrast, crosslinking of CD81 on TCRalphabeta(+) T cells, in the absence of anti-CD3 stimulation, failed to activate these T cells. These results suggest that CD81 signaling may be mediated via a different mechanism(s) in TCRgammadelta(+) versus TCRalphabeta(+) T cells.

Stem cell factor and IL-2 act synergistically in inducing intraepithelial lymphocyte proliferation and cytokine production: upregulation of the IL-2 receptor gamma-chain and signaling via JAK-3.

Murine intraepithelial lymphocytes (IEL) that express the gamma/delta form of the T cell receptor for antigen (TCRgammadelta) also express c-kit, the receptor for stem cell factor (SCF). We show here that SCF upregulates the expression of gammadelta TCR on IEL. More importantly, SCF induces upregulation in the expression of the common gamma-chain (gammac), which is a shared subunit of the receptor complexes for IL-2, -4, -7, -9, and -15. SCF was shown to act synergistically with IL-2 in inducing IEL proliferation, IFNgamma production, non-MHC-restricted cytotoxic activity, and upregulation of the expression of the gammac. SCF also acted synergistically with IL-7 and IL-15 in inducing IEL proliferation. IEL exposed to SCF were shown to have enhanced phosphorylation of JAK-3, and when SCF was combined with IL-2, there was an enhancement in the phosphorylation of JAK-3. These results suggest that SCF may play a more important role in regulating mucosal immune responses than previously appreciated.

Stem cell factor (SCF) can regulate the activation and expansion of murine intraepithelial lymphocytes.

Murine intraepithelial lymphocytes (IEL) express c-kit, the receptor for stem cell factor (SCF). SCF induced a low but significant proliferative response in IEL, but not in splenic T cells. SCF stimulation of IEL resulted in an expansion of the c-kit(+), TCRgammadelta(+)cell population. SCF-induced proliferation was dependent upon SCF-c-kit interactions, since antibody to c-kit blocked this response, and IEL obtained from c-kit mutant (W/W(v)) mice failed to respond to SCF. SCF acted synergistically with anti-TCRgammadelta and with concavalin A (Con A) to induce proliferation and interferon gamma (IFN-gamma) production in IEL. Finally, mice injected with SCF had a significant increase in the number of IEL in the small intestine. SCF-treated mice had increased numbers of TCRalphabeta(+)and TCRgammadelta(+)cell populations, as well as increased numbers of c-kit(+)and c-kit(-)IEL. These data suggest that SCF-c-kit interactions play an important role in regulating IEL expansion and activation.

Effect of CYP2E1 induction by ethanol on the immunotoxicity and genotoxicity of extended low-level benzene exposure.

Potential additive effects of ethanol consumption, a common life-style factor, and low-level benzene exposure, a ubiquitous environmental pollutant, were investigated. Ethanol is a potent inducer of the cytochrome P-450 2E1 (CYP2E1) enzyme, which bioactivates benzene to metabolites with known genotoxicity and immunotoxicity. A liquid diet containing 4.1% ethanol was used to induce hepatic CYP2E1 activity by 4-fold in female CD-1 mice. Groups of ethanol-treated or pair-fed control mice were exposed to benzene or filtered air in inhalation chambers for 7 h/d, 5 d/wk for 6 or 11 wk. The initial experiment focused on immunotoxicity endpoints based on literature reports that ethanol enhances high-dose benzene effects on spleen, thymus, and bone marrow cellularity and on peripheral red blood cell (RBC) and white blood cell (WBC) counts. No statistically significant alterations were found in spleen lymphocyte cellularity, subtype profile, or function (mitogen-induced proliferation, cytokine production, or natural killer cell lytic activity) after 6 wk of ethanol diet, 0.44 ppm benzene exposure, or both. This observed absence of immunomodulation by ethanol alone, a potential confounding factor, further validates our previously established murine model of sustained CYP2E1 induction by dietary ethanol. Subsequent experiments involved a 10-fold higher benzene level for a longer time of 11 wk and focused on genotoxic endpoints in known target tissues. Bone marrow and spleen cells were evaluated for DNA-protein cross-links, a sensitive transient index of genetic damage, and spleen lymphocytes were monitored for hprt-mutant frequency, a biomarker of cumulative genetic insult. No treatment-associated changes in either genotoxic endpoint were detected in animals exposed to 4.4 ppm benzene for 6 or 11 wk with or without coexposure to ethanol. Thus, our observations suggest an absence of genetic toxicity in CD-1 mice exposed to environmentally relevant levels of benzene with or without CYP2E1 induction.

Expression of IL-10 receptors on epithelial cells from the murine small and large intestine.

The appearance of chronic intestinal inflammation in IL-10 knockout mice suggests IL-10 may inhibit adverse responses to luminal antigen. Moreover, this inflammation is associated with an increase in class II MHC molecule expression on intestinal epithelial cells. Thus, the role of IL-10 regulation in epithelial cell function was investigated. Using RT-PCR, it was shown that intestinal epithelial cells express mRNA for both subunits of the IL-10 receptor-signaling complex. In addition, biotinylated IL-10 was shown to bind to both cultured and freshly isolated intestinal epithelial cells prepared from the small or large intestine. This binding appeared specific as it was blocked by neutralizing antibodies to IL-10 but not the isotype control. Moreover, an excess of native IL-10 also inhibited the binding of radiolabeled IL-10. To evaluate whether IL-10 mediated any functions through this receptor, epithelial cells were cultured with IL-10 alone or with IFN-gamma plus IL-10. IL-10 alone had no detectable effects on epithelial cell growth or their expression of class II MHC molecules but it did antagonize the effect of IFN-gamma on the viability of cultured cells. In addition, IL-10 blocked the IFN-gamma-induced expression of class II MHC molecules on cultured epithelial cells. These results suggest that IL-10 binds to a specific receptor on intestinal epithelial cells and may regulate the contribution of epithelial cells to the inflammatory and immune response in the digestive tract.

Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14.

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.

Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.

Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor-2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor-kappaB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms.

Regulation of the mucosal immune response.

Infectious diseases continue to exact an extensive toll on populations living closest to the equatorial regions of the globe. A substantial proportion of these infections gain access to the host via the mucosal tissues. Thus, the development of new vaccines that enhance mucosal immunity is considered to be of paramount importance in order to prevent or limit the impact of these infections. Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive amounts of inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the unique properties of mucosal immune responses and focus on recent advances that have significantly influenced our understanding of the regulation of immune and inflammatory responses following infection.

Lipoprotein release by bacteria: potential factor in bacterial pathogenesis.

Lipoprotein (LP) is a major component of the outer membrane of bacteria in the family Enterobacteriaceae. LP induces proinflammatory cytokine production in macrophages and lethal shock in LPS-responsive and -nonresponsive mice. In this study, the release of LP from growing bacteria was investigated by immuno-dot blot analysis. An immuno-dot blot assay that could detect LP at levels as low as 100 ng/ml was developed. By using this assay, significant levels of LP were detected in culture supernatants of growing Escherichia coli cells. During mid-logarithmic growth, approximately 1 to 1.5 microgram of LP per ml was detected in culture supernatants from E. coli. In contrast, these culture supernatants contained 5 to 6 microgram/ml of lipopolysaccharide (LPS). LP release was not unique to E. coli. Salmonella typhimurium, Yersinia enterocolitica, and two pathogenic E. coli strains also released LP during in vitro growth. Treatment of bacteria with the antibiotic ceftazidime significantly enhanced LP release. Culture supernatants from 5-h cultures of E. coli were shown to induce in vitro production of interleukin-6 (IL-6) by macrophages obtained from LPS-nonresponsive C3H/HeJ mice. In contrast, culture supernatants from an E. coli LP-deletion mutant were significantly less efficient at inducing IL-6 production in C3H/HeJ macrophages. These results suggest, for the first time, that LP is released from growing bacteria and that this released LP may play an important role in the induction of cytokine production and pathologic changes associated with gram-negative bacterial infections.

Apoptotic killing of CD4+ T lymphocytes in HIV-1-infected PHA-stimulated PBL cultures is mediated by CD8+ LAK cells.

In vitro infection of PHA-stimulated, normal CD4+ human peripheral blood T lymphocytes (PBLs) with several HIV-1 isolates did not result in cytopathology, despite high levels of virus replication and the fact that some of these isolates were cytopathic in certain cell lines. In contrast, infection of unfractionated PBLs (containing CD8+ as well as CD4+ lymphocytes) with these isolates always resulted in death of the infected CD4+ T lymphocytes. It has been well documented that PHA stimulation and culture of PBLs in medium containing IL-2 generates lymphokine-activated killer (LAK) cell activity which can destroy many transformed cells and virus-infected normal cells. When CD8+ T lymphocytes from PHA-stimulated PBLs were added to HIV-1-infected purified CD4+ T lymphocytes, significant lysis occurred. This cytotoxicity was not MHC class I-restricted, and depletion of CD8+ T lymphocytes from unfractionated PBL cultures shortly after HIV infection largely abolished the killing of the infected CD4+ T lymphocytes. These results demonstrated that CD8+ LAK cells were killing the CD4+ T lymphocytes in unfractionated PBL cultures infected with these noncytopathic HIV-1 strains. Care is thus warranted when studying HIV cytopathology in unfractionated PBL cultures. Morphological and DNA gel electrophoretic analyses of HIV-infected CD4+ T lymphocytes being killed by CD8+ LAK cells demonstrated that apoptosis was the predominant mechanism of LAK cell-mediated killing. In contrast, necrosis was the major mechanism involved in killing of purified CD4+ T lymphocytes by HIV-1 strains which were directly cytopathic. These findings may explain some of the discrepancies in the literature concerning reports of either apoptotic or necrotic killing of cells by HIV in vitro. Moreover, these data strongly suggest that direct killing by replicating HIV-1 in vivo should reveal necrotic cells and immune effector cell killing should reveal apoptotic cells. Since the latter are much more frequently observed in vivo, perhaps immune effector-mediated depletion of CD4+ T lymphocytes is more important as a pathogenic mechanism.

Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production.

Septic shock is a major cause of death in the world. Although much is known about the role of LPS in septic shock, little is known about the role of other bacterial components. Lipoprotein (LP) is a major component of bacteria in the family Enterobacteriaceae. LP purified from Escherichia coli was shown to induce TNF-alpha and IL-6 production in peritoneal exudate macrophages obtained from LPS-responsive (C3H/HeOuJ) and LPS-nonresponsive (C3H/HeJ) mice. LP and LPS acted synergistically to induce cytokine production not only in C3H/HeOuJ macrophages but also in C3H/HeJ macrophages. These results suggest that LPS can induce cellular signaling in C3H/HeJ macrophages, and that LPS and LP activate macrophages via different receptors and/or signaling pathways. The role LP plays in septic shock was investigated using the mouse D-galactosamine model. LP induced lethal shock and in vivo production of TNF-alpha and IL-6 in both LPS-responsive and LPS-nonresponsive mice. LPS failed to induce lethal shock or in vivo cytokine production in C3H/HeJ mice. However, LP and LPS acted synergistically in inducing lethal shock and in vivo cytokine production in both LPS-responsive and LPS-nonresponsive mice. Finally, a heat-killed preparation of an E. coli mutant strain that lacked LP was shown to be less efficient than heat-killed wild-type E. coli at inducing lethal shock in C3H/HeJ mice. Collectively, these results suggest that LP and LPS induce cytokine production via different mechanisms and that LP plays an important role in septic shock induced by bacteria in the family Enterobacteriaceae.

Lipoprotein from Yersinia enterocolitica contains epitopes that cross-react with the human thyrotropin receptor.

Yersinia enterocolitica has recently been shown to produce a low molecular mass envelope protein that contains an epitope(s) that is cross-reactive with the extracellular domain of the human thyrotropin receptor (ETSHR). In this study, we have generated mAb to this cross-reactive protein and have obtained amino acid sequences for peptide fragments obtained from Lys-c digestion of the protein. The amino acid sequences of these peptides were identical to sequences present in bacterial lipoprotein (LP). All bacteria of the Enterobacteriaceae family produce LP as a major outer membrane protein. However, the ETSHR cross-reactive epitope(s) was shown to be unique to LP produced by Yersinia species. This was shown by Western blot analysis using a mAb specific for LP and with affinity-purified Ab specific for either LP or ETSHR and obtained from mouse antiserum generated to Y. enterocolitica. LPs from different Gram-negative bacteria were shown to be mitogenic for C3H/HeJ spleen cells and induced production and secretion of significant levels of Ig. Production of Ab that recognized the ETSHR was only induced in spleen cells stimulated with the LP obtained from Yersinia. In contrast, LP was not mitogenic for either human PBMC or human B cells. However, LP did induce IL6 and IL8 production in human monocytes at levels equivalent to that seen after LPS activation. These results identify, for the first time, the Yersinia envelope protein that is cross-reactive with the ETSHR and show that it can activate human monocytes. These findings are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the thyrotropin receptor.

Phospholipase A2 activating protein and idiopathic inflammatory bowel disease.

BACKGROUND: Crohn's disease and ulcerative colitis are idiopathic inflammatory bowel diseases (IBD) involving synthesis of eicosanoids from arachidonic acid (AA), which is released from membrane phospholipids by phospholipase A2 (PLA2). A potentially important regulator of the production of these mediators is a protein activator of PLA2, referred to as PLA2 activating protein (PLAP). AIMS: The purpose of this investigation was to discover if PLAP values might be increased in the inflamed intestinal tissue of patients with IBD and in intestinal tissue of mice with colitis. PATIENTS: Biopsy specimens were taken from patients with ulcerative colitis and Crohn's disease undergoing diagnostic colonoscopy, and normal colonic mucosa was obtained from patients without IBD after surgical resection. METHODS: Immunocytochemistry with affinity purified antibodies to PLAP synthetic peptides was used to locate PLAP antigen in sections of intestinal biopsy specimens from IBD patients compared with that of normal intestinal tissue. Northern blot analysis with a murine [32P] labelled plap cDNA probe was performed on RNA extracted from the colons of mice fed dextran sulphate sodium (DSS) and cultured HT-29 cells exposed to lipopolysaccharide (LPS). RESULTS: PLAP antigen was localised predominantly within monocytes and granulocytes in intestinal tissue sections from IBD patients, and additional deposition of extracellular PLAP antigen was associated with blood vessels and oedema fluid in the inflamed tissues. In contrast, tissue sections from normal human intestine were devoid of PLAP reactive antigen, except for some weak cytoplasmic reaction of luminal intestinal epithelial cells. Similarly, colonic tissue from DSS treated mice contained an increased amount of PLAP antigen compared with controls. The stroma of the lamina propria of the colonic mucosa from the DSS treated mice reacted intensely with antibodies to PLAP synthetic peptides, while no reaction was observed with control mouse colons. These data were supported by northern analysis which showed that PLAP mRNA was increased in the colons of DSS treated mice and cultured HT-29 cells exposed to LPS. CONCLUSIONS: As PLAP values were increased in the intestinal mucosa of IBD patients and mice with colitis, as well as in LPS treated cultured HT-29 cells, a role was postulated for PLAP in increasing PLA2 activity, which leads to the increased synthesis of eicosanoids in intestinal tissues of patients with these inflammatory diseases.

Yersinia enterocolitica envelope proteins that are crossreactive with the thyrotropin receptor (TSHR) also have B-cell mitogenic activity.

Autoantibodies to the thyrotropin receptor (TSHR) have been shown to mediate the hyperthyroidism associated with Graves' disease (GD). A number of hypotheses have been proposed which link an infectious agent to the mechanism(s) involved in the induction of GD. Several studies have suggested that the development of GD may be linked to infection with the enteric pathogen Yersinia enterocolitica. We have recently identified two low molecular weight (5.5 and 8 kDa) envelope proteins of Y. enterocolitica that are cross-reactive with the extracellular domain of human TSHR (ETSHR). In this study, we have purified these ETSHR-crossreactive Yersinia proteins (TSHR-CRP) and have further characterized their immunoreactivity. Both the 5.5 and 8 kDa TSHR-CRPs were shown to be mitogenic for mouse spleen cells. This mitogenic activity was specific for B cells and was not due to lipopolysaccharide (LPS) contamination. TSHR-CRPs were mitogenic for LPS-non-responsive spleen cells obtained from C3H/Hej mice, and polymyxin B did not inhibit the mitogenic activity of the TSHR-CRPs. TSHR-CRPs also induced high levels of IL-6 production in B cells and induced production and secretion of significant levels of IgG and IgM. Finally, culture supernatants from TSHR-CRP-stimulated spleen cells were shown by Western blot analysis to contain antibodies that recognized the ETSHR These results identify for the first time two envelope proteins of Yersinia that have mitogenic activity and therefore could represent important proteins involved in the pathogenesis of Yersinia infections. Because these mitogenic proteins also contain epitopes crossreactive with the TSHR, they are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the TSHR.