Allen J. Rosenspire

Cutting Edge: Fever-Associated Temperatures Enhance Neutrophil Responses to Lipopolysaccharide: A Potential Mechanism Involving Cell Metabolism

Although much progress has been made in elucidating the mechanisms underlying the physiological regulation of fever, there is little understanding of the biological utility of fever’s thermal component. Considering the evolutionary co-conservation of fever and innate immunity, we hypothesize that fever’s thermal component might in general augment innate immune function and, in particular, neutrophil activation. Accordingly, we have evaluated the effect of febrile temperatures on neutrophil function at the single-cell level. We find that reactive oxygen intermediates and NO release are greatly enhanced at febrile temperatures for unstimulated as well as LPS-stimulated adherent human neutrophils. Furthermore, our studies suggest that these changes in oxidant release are linked to upstream changes in NADPH dynamics. Inasmuch as reactive oxygen intermediates and NO production are important elements in innate immune responses to bacterial pathogens, we suggest that the febrile rise in core temperature is a broad-based systemic signaling mechanism to enhance innate immunity.

Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis

Non-porous reversed-phase (NP-RP) HPLC has been used to rapidly generate protein profiles of whole cell lysates of human breast cancer cell lines. The non-porous packing material used was silica coated with C18, which provided rapid separation with high collection efficiency of proteins from cell lysates. This method was used to study the differences in protein profiles among normal cells and fully malignant cells that share a common genetic background. The highly expressed proteins in each cell type were separated and collected in the liquid state where they were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) to obtain the molecular weight of the proteins. The protein fractions were then subjected to tryptic digestion and analyzed by pulsed delay extraction (PDE)-MALDI-TOFMS to obtain the peptide maps. The expressed proteins were identified based upon the molecular weight and peptide map using database-searching procedures. It is shown that key cancer-related proteins can be detected and identified which may be potentially used as biomarkers for cancer detection.

Protein Profiles and Identification of High Performance Liquid Chromatography Isolated Proteins of Cancer Cell Lines Using Matrix- assisted laser desorption/ionization Time-of-flight Mass Spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.

HLA Immune Function Genes in Autism

The human leukocyte antigen (HLA) genes on chromosome 6 are instrumental in many innate and adaptive immune responses. The HLA genes/haplotypes can also be involved in immune dysfunction and autoimmune diseases. It is now becoming apparent that many of the non-antigen-presenting HLA genes make significant contributions to autoimmune diseases. Interestingly, it has been reported that autism subjects often have associations with HLA genes/haplotypes, suggesting an underlying dysregulation of the immune system mediated by HLA genes. Genetic studies have only succeeded in identifying autism-causing genes in a small number of subjects suggesting that the genome has not been adequately interrogated. Close examination of the HLA region in autism has been relatively ignored, largely due to extraordinary genetic complexity. It is our proposition that genetic polymorphisms in the HLA region, especially in the non-antigen-presenting regions, may be important in the etiology of autism in certain subjects.

Inorganic mercury attenuates CD95-mediated apoptosis by interfering with formation of the death inducing signaling complex.

Inorganic mercury (Hg2+) modulates several lymphocyte signaling pathways and has been implicated as an environmental factor linked to autoimmune disease. From the standpoint that autoimmune diseases represent disorders of cell accumulation, in which dysregulated apoptosis may be one mechanism leading to the accumulation of autoreactive lymphocytes, we have been investigating the influences of Hg2+ on CD95-mediated apoptosis. We demonstrate here that low and noncytotoxic concentrations of Hg2+ impair CD95 agonist-induced apoptosis in representative Type-I and Type-II T cell lines. Hg2+ treatment blocks the CD95 agonist-induced activation of initiator and effector caspases as well as the association between CD95 and the signaling adaptor, FADD. CD95 multimerization does not appear to be affected by Hg2+. Thus, the Hg2+ sensitive step within the CD95 death pathway is localized to the level of the death inducing signaling complex (DISC). Disruption of proper DISC formation may be a biochemical mechanism whereby Hg2+ contributes to autoimmune disease.

Mercury Inhibition of Neutrophil Activity: Evidence of Aberrant Cellular Signalling and Incoherent Cellular Metabolism

Exposure to environmental heavy metals has been reported to affect the immune system. Here, we tested the hypothesis that Hg+2, acting through membrane proteins, disrupts metabolic dynamics and downstream cell functions in human neutrophils. We found that HgCl2 inhibited: (1) polarization and (2) immunoglobulin (Ig)G‐mediated phagocytosis of sheep erythrocytes in a dose‐dependent manner from 2.5 to 10 µm. Because these activities have been linked with pro‐inflammatory signalling, we also studied the effects of HgCl2 on intracellular signalling by measuring protein tyrosine phosphorylation. HgCl2 at doses = 1 µm increased tyrosine phosphorylation. We also studied the effect of HgCl2 on neutrophil metabolism by measuring NAD(P)H autofluorescence as an indicator of intracellular NAD(P)H concentration. After HgCl2 treatment, we found that normal sinusoidal NAD(P)H oscillations became incoherent. We recently reported that the NAD(P)H oscillation frequency is affected by cell migration and activation, which can in turn be regulated by integrin‐mediated signalling. Therefore, we examined the effects of HgCl2 on cell surface distribution of membrane proteins. After exposure to environmentally relevant concentrations of HgCl2 we found that CR3, but not other membrane proteins (e.g. uPAR, FcγRIIA and the formyl peptide receptor), became clustered on cell surfaces. We suggest that HgCl2 disrupts integrin signalling/functional pathways in neutrophils.

Interferon-γ and sinusoidal electric fields signal by modulating NAD(P)H oscillations in polarized neutrophils

Metabolic activity in eukaryotic cells is known to naturally oscillate. We have recently observed a 20-s period NAD(P)H oscillation in neutrophils and other polarized cells. Here we show that when polarized human neutrophils are exposed to interferon-gamma or to ultra-low-frequency electric fields with periods double that of the NAD(P)H oscillation, the amplitude of the NAD(P)H oscillations increases. Furthermore, increases in NAD(P)H amplitude, whether mediated by interferon-gamma or by an oscillating electric field, signals increased production of reactive oxygen metabolites. Hence, amplitude modulation of NAD(P)H oscillations suggests a novel signaling mechanism in polarized cells.

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.

CD4-Independent inhibition of lymphocyte proliferation mediated by HIV-1 envelope glycoproteins

The cytopathic effects of HIV-1 produced by direct infection of human T cells do not account for the disproportionate loss of CD4-positive lymphocytes during the course of HIV infection. Previous studies have demonstrated the inhibition of uninfected human T cell activation and proliferation by the HIV-1 envelope glycoproteins, presumably due to gp120-CD4 interactions. To examine the ability of HIV-1 to inhibit T cell proliferation in the absence of both direct infection and gp120-CD4 interactions, we tested the effect of HIV-1 on mouse T cell proliferation. Culture media containing HIV-1 released from infected cells inhibited T lymphocyte proliferation in response to interleukin-2 (IL-2). Studies to explore the mechanism of this inhibition suggested that the decrease in proliferation resulted from interactions between HIV-1 and the mouse cells, but did not involve IL-2/IL-2 receptor interactions. We used monoclonal antibodies to demonstrate that the HIV-1 envelope glycoproteins were required for the inhibition of murine T cell proliferation. Anti-gp120 antibodies completely restored proliferation, indicating that the surface protein gp120 was primarily required for the inhibition of proliferation. However, antibodies directed against the transmembrane protein of HIV-1 (gp41) also partially restored lymphocyte proliferation. The functional significance of the HIV-1 envelope protein epitopes recognized by the monoclonal antibodies is discussed.

T-cell receptor signaling is mediated by transient Lck activity, which is inhibited by inorganic mercury

Genetically susceptible rodents exposed to low nontoxic levels of inorganic mercury (Hg2+) develop idiosyncratic autoimmune disease associated with defective T‐cell function. However, the molecular mechanisms underlying this phenomenon remain mostly unexplained. Brief exposure of T cells to micromolar concentrations of Hg2+ leads to physiologically relevant nontoxic cellular mercury burdens, and as we have previously reported, attenuates T‐cell receptor (TCR) signal strength by ~50%. We have found this to be the result of an inadequate activation of the tyrosine kinase ZAP‐70, which is hypophosphorylated following TCR stimulation in Hg2+ burdened cells when compared to untreated controls. In T cells,ZAP‐70 phosphorylation is dependent on lymphocyte‐specific protein tyrosine kinase (Lck) activity, which in turn is either positively or negatively regulated by the phosphorylation of specific Lck tyrosine residues. In particular, the general belief is that Lck is negatively regulated by phosphorylation of tyrosine 192 (Y192). We now demonstrate by Western blotting that, in Jurkat T cells, TCR signal transduction (andZAP‐70 phosphorylation) was positively associated with a rapid transient phosphorylation of Y192, which was inhibited in cells that were briefly (5 min) exposed to 5 μM Hg2+. Thus, Hg2+ inhibits a critical activating role played by Lck Y192 during the most proximal events of the TCR‐induced cell signaling.—Ziemba, S.E., Menard, S.L., McCabe, Jr., M.J., Rosenspire, A.J. T‐cell receptor signaling is mediated by transient Lck activity, which is inhibited by inorganic mercury. FASEB J. 23, 1663–1671 (2009)