Randall F. Gill

Systematic identification of H-2 Kd binding peptides and induction of peptide specific CTL.

Most peptides with putative MHC I restricted sequence motifs do not bind to the corresponding MHC I nor induce cytolytic T cells. There exist additional constraints which limit peptide binding and immunogenicity. To identify immunogenic peptides in novel protein sequences, it will be necessary to first evaluate peptide binding to MHC I. In this study, a soluble single chain fusion protein SC-Kd was used to evaluate potential Kd binding peptides from the sequences of mouse mammary tumor virus gag and env proteins. A total of 27 peptides were identified which displayed the reported Kd restricted motif. Of the 27 peptides, six demonstrated strong to moderate binding to SC-Kd. The strongest binding peptides expressed tyrosine or phenylalanine at position 2 and leucine at the C-terminus. The capability of MMTV peptides to induce CTL corresponds to their SC-Kd binding activity. Of the six peptides that demonstrated moderate to strong binding, five induced CTL in BALB/c mice. These peptides induced CTL after 1-3 in vivo immunizations followed by 5 day in vitro stimulation. Furthermore, a single in vitro stimulation of naive lymphocytes with strong-binding G425 was sufficient to induce significant CTL activity. Weak or non-binding peptides did not induce CTL. Therefore, peptide binding to SC-Kd is a predictive indicator of CTL inducing activity.

A systems toxicology approach identifies Lyn as a key signaling phosphoprotein modulated by mercury in a B lymphocyte cell model

Network and protein-protein interaction analyses of proteins undergoing Hg²⁺-induced phosphorylation and dephosphorylation in Hg²⁺-intoxicated mouse WEHI-231 B cells identified Lyn as the most interconnected node. Lyn is a Src family protein tyrosine kinase known to be intimately involved in the B cell receptor (BCR) signaling pathway. Under normal signaling conditions the tyrosine kinase activity of Lyn is controlled by phosphorylation, primarily of two well known canonical regulatory tyrosine sites, Y-397 and Y-508. However, Lyn has several tyrosine residues that have not yet been determined to play a major role under normal signaling conditions, but are potentially important sites for phosphorylation following mercury exposure. In order to determine how Hg²⁺ exposure modulates the phosphorylation of additional residues in Lyn, a targeted MS assay was developed. Initial mass spectrometric surveys of purified Lyn identified 7 phosphorylated tyrosine residues. A quantitative assay was developed from these results using the multiple reaction monitoring (MRM) strategy. WEHI-231 cells were treated with Hg²⁺, pervanadate (a phosphatase inhibitor), or anti-Ig antibody (to stimulate the BCR). Results from these studies showed that the phosphoproteomic profile of Lyn after exposure of the WEHI-231 cells to a low concentration of Hg²⁺ closely resembled that of anti-Ig antibody stimulation, whereas exposure to higher concentrations of Hg²⁺ led to increases in the phosphorylation of Y-193/Y-194, Y-501 and Y-508 residues. These data indicate that mercury can disrupt a key regulatory signal transduction pathway in B cells and point to phospho-Lyn as a potential biomarker for mercury exposure.

Enhancement of rat tear IgA antibody responses following intranasal immunization with antigen and CpG ODN.

Purpose. To determine the effect of unmethylated oligodeoxynucleotides containing bacterial CpG motifs (CpG ODN) on the induction of rat tear IgA antibody responses. Methods. Rats were immunized intranasally with either soluble dinitrophenylated bovine serum albumin (DNP-BSA) or poly(lactide-co-glycolide) (PLG) encapsulated DNP-BSA in combination with CpG ODN. The animals received two immunizations 21 days apart. Following the second immunization, tear, saliva and serum samples were collected for 28 days and analyzed for antigen specific antibodies. Tear IgA, saliva IgA and serum IgG antibody concentrations were determined by ELISA. Results. Co-administration of CpG ODN with either soluble or encapsulated antigen resulted in significantly elevated levels of both tear and salivary IgA antibodies as well as levels of serum IgG antibodies. Microencapsulated DNP-BSA plus CpG ODN elicited higher levels of IgA antibodies in tears than did soluble antigen plus CpG ODN. Conclusions. CpG ODN is an effective mucosal immune modulator for enhancing rat tear IgA antibody responses to both soluble and microencapsulated antigens.

Nasal-associated lymphoid tissue is not an absolute requirement for the induction of rat tear IgA antibody responses.

Purpose/Aim of study: The purpose of this work was to determine whether rat nasal-associated lymphoid tissue is required for the induction of tear IgA responses. Materials and Methods: Particulate antigen in the form of DNP-BSA encapsulated in cationic microparticles was applied topically to the eyes (ocular topically) of rats that had the nasolacrimal ducts temporarily plugged with chromic gut suture material. Eye washes and serum were monitored for development of antigen specific IgA and IgG, respectively. To track the particulate uptake, fluorescent latex beads were applied topically to the eyes of plugged and unplugged animals. The nasal-associated lymphoid tissue and the draining lymph nodes were then examined for the presence of the fluorescent beads. Results: It was found that the chromic gut suture was effective in blocking the passage of antigen into the nasopharyngeal cavity for at least 24 hr. Tear antigen-specific IgA levels found in the eyes of plugged animals were not significantly lower from those of unplugged animals. Serum IgG antibody levels were also similar between the two groups. In animals with plugged nasolacrimal ducts, fluorescent beads were found predominately in the superficial cervical lymph nodes, which have been shown to drain the surface of the eye. Conclusions: These results indicate that particulate antigen can be taken up by the conjunctiva and transported to the draining lymph nodes, showing that antigen does not need to access nasal-associated lymphoid tissue to induce tear IgA antibody responses.

Elements of the B Cell Signalosome Are Differentially Affected by Mercury Intoxication

It has been suggested that environmental exposures to mercury contribute to autoimmune disease. Disruption of BCR signaling is associated with failure of central tolerance and autoimmunity, and we have previously shown that low levels of Hg2+ interfere with BCR signaling. In this report we have employed multiparametric phosphoflow cytometry, as well as a novel generalization of the Overton algorithm from one- to two-dimensional unimodal distributions to simultaneously monitor the effect of low level Hg2+ intoxication on activation of ERK and several upstream elements of the BCR signaling pathway in WEHI-231 B cells. We have found that, after exposure to low levels of Hg2+, only about a third of the cells are sensitive to the metal. For those cells which are sensitive, we confirm our earlier work that activation of ERK is attenuated but now report that Hg2+ has little upstream effect on the Btk tyrosine kinase. On the other hand, we find that signaling upstream through the Syk tyrosine kinase is actually augmented, as is upstream activation of the B cell signalosome scaffolding protein BLNK.

Inductive Sites for Rat Tear IgA Antibody Responses

The mucosal immune system is an important mediator of acquired immunity at the ocular surface.1,2 In mammals, the lacrimal gland serves as the predominant source of tear IgA3,4 and functions as an effector site for mucosal antibody responses.1,2 Evidence for the linkage of lacrimal glands to the mucosal network has come from cell trafficking5,6 as well as antibody expression data.7–9 In a number of species, oral or gastrointestinal antigen stimulation has resulted in tear IgA antibody induction.7–12 In addition, topical application of particulate antigen7–9 or microparticle-encapsulated soluble antigen13 to the conjunctiva (ocular topical, OT) has been documented to be an effective means of eliciting tear IgA antibody responses. Furthermore, evidence indicates that nasal-associated lymphoid tissue (NALT) is a major inductive site in the mucosal immune network,14,15 and NALT appears to serve as an inductive site for tear IgA antibody responses.16

Docosahexaenoic acid counteracts attenuation of CD95-induced cell death by inorganic mercury

In the United States the principal environmental exposure to mercury is through dietary consumption of sea food. Although the mechanism by which low levels of mercury affect the nervous system is not well established, epidemiological studies suggest that low level exposure of pregnant women to dietary mercury can adversely impact cognitive development in their children, but that Docosahexaenoic acid (DHA), the most prominent n-polyunsaturated fatty acid (n-PUFA) present in fish may counteract negative effects of mercury on the nervous system. Aside from effects on the nervous system, epidemiological and animal studies have also suggested that low level mercury exposure may be a risk factor for autoimmune disease. However unlike the nervous system where a mechanism linking mercury to impaired cognitive development remains elusive, we have previously suggested a potential mechanism linking low level mercury exposures to immune system dysfunction and autoimmunity. In the immune system it is well established that disruption of CD95 mediated apoptosis leads to autoimmune disease. We have previously shown in vitro as well as in vivo that in lymphocytes burdened with low levels of mercury, CD95 mediated cell death is impaired. In this report we now show that DHA counteracts the negative effect of mercury on CD95 signaling in T lymphocytes. T cells which have been pre-exposed to DHA are able to cleave pro-caspase 3 and efficiently signal programmed cell death through the CD95 signaling pathway, whether or not they are burdened with low levels of mercury. Thus DHA may lower the risk of autoimmune disease after low level mercury exposures.

Low level exposure to inorganic mercury interferes with B cell receptor signaling in transitional type 1 B cells

ABSTRACT Mercury (Hg) has been implicated as a factor contributing to autoimmune disease in animal models and humans. However the mechanism by which this occurs has remained elusive. Since the discovery of B cells it has been appreciated by immunologists that during the normal course of B cell development, some immature B cells must be generated that produce immunoglobulin reactive to self‐antigens (auto‐antibodies). However in the course of normal development, the vast majority of immature auto‐reactive B cells are prevented from maturing by processes collectively known as tolerance. Autoimmune disease arises when these mechanisms of tolerance are disrupted. In the B cell compartment, it is firmly established that tolerance depends in part upon negative selection of self‐reactive immature (transitional type 1) B cells. In these cells negative selection depends upon signals generated by the B Cell Receptor (BCR), in the sense that those T1 B cells whos BCRs most strongly bind to, and so generate the strongest signals to self‐antigens are neutralized. In this report we have utilized multicolor phosphoflow cytometry to show that in immature T1 B cells Hg attenuates signal generation by the BCR through mechanisms that may involve Lyn, a key tyrosine kinase in the BCR signal transduction pathway. We suggest that exposure to low, environmentally relevant levels of Hg, disrupts tolerance by interfering with BCR signaling in immature B cells, potentially leading to the appearance of mature auto‐reactive B cells which have the ability to contribute to auto‐immune disease. HIGHLIGHTSHg2 + interferes with BCR‐mediated activation of ERK in immature T1 B cells.Hg2 + acts upstream of ERK.BCR mediated activation of Syk and the CD79a ITAM are attenuated in immature T1 B cells by Hg2 +.The mechanism whereby which Hg2 + affects Syk and CD7a activation involves the tyrosine phosphokinase Lyn.

Dietary n-3 PUFAs augment caspase 8 activation in Staphylococcal aureus enterotoxin B stimulated T-cells.

Epidemiological studies have linked consumption of n-3 PUFAs with a variety of beneficial health benefits, particularly with respect to putative anti-inflammatory effects. Unfortunately, many of these results remain somewhat controversial because in most instances there has not been a linkage to specific molecular mechanisms. For instance, dietary exposure to low levels of mercury has been shown to be damaging to neural development, but concomitant ingestion of n-3 PUFAs as occurs during consumption of fish, has been shown to counteract the detrimental effects. As the mechanisms mediating the neurotoxicity of environmental mercury are not fully delineated, it is difficult to conceptualize a testable molecular mechanism explaining how n-3 PUFAs negate its neurotoxic effects. However, environmental exposure to mercury also has been linked to increased autoimmunity. By way of a molecular understanding of this immuno-toxic association, disruption of CD95 signaling is well established as a triggering factor for autoimmunity, and we have previously shown that environmentally relevant in vitro and dietary exposures to mercury interfere with CD95 signaling. In particular we have shown that activation of caspase 8, as well as downstream activation of caspase 3, in response to CD95 agonist stimulation is depressed by mercury. More recently we have shown in vitro that the n-3 PUFA docosahexaenoic acid counteracts the negative effect of mercury on CD95 signaling by restoring caspase activity. We hypothesized that concomitant ingestion of n-3 PUFAs with mercury might be protective from the immuno-toxic effects of mercury, as it is with mercurys neuro-toxic effects, and in the case of immuno-toxicity this would be related to restoration of CD95 signal strength. We now show that dietary ingestion of n-3 PUFAs generally promotes CD95 signaling by upregulating caspase 8 activation. Apart from accounting for the ability of n-3 PUFAs to specifically counteract autoimmune sequelae of mercury exposure, this novel finding for the first time suggests a testable molecular mechanism explaining the overall anti-inflammatory properties of n-3 PUFAs.