Papers by Thomas Hamilton
Journal of Immunology, Feb 1, 1989
It has been previously demonstrated that maley-lated-BSA (maleyl-albumin) induces functional acti... more It has been previously demonstrated that maley-lated-BSA (maleyl-albumin) induces functional activation in murine peritoneal macrophages. Furthermore, maleyl-albumin has been shown to interact with two distinct sites on human monocytes; one site is the scavenger receptor, a 260-kDa oligomeric protein which recognizes modified forms of low density lipoprotein (LDL), and the second is a lower affinity site which has yet to be structurally characterized. In the present study, we wished to quantitatively assess the number and character of maleyl-albumin-binding sites on murine peritoneal macrophages and to determine which site or sites are involved in signaling the macrophage to undergo extensive functional development. Binding studies. demonstrate at least two distinct receptors for maleyl-albumin on murine peritoneal macrophages. Scatchard analyses of the binding isotherms reveal two sites characterized by dissociation constants (Kd) of 17.6 nM and 4.9 microM and maximal binding of 1.2 x 10(5) and 1 x 10(6) sites/cell, respectively. The contribution of the scavenger receptor, determined by binding analyses of malondialdehyde-LDL, is described by two sites with Kd of 39.4 pM and 9.6 nM, and maximal binding of 2.7 x 10(3) and 1.9 x 10(4) sites/cell, respectively. Maleyl-albumin blocks binding of malondialdehyde-LDL, whereas modified LDL fails to inhibit binding of maleyl-albumin. Maleyl-albumin, at concentrations producing lower affinity binding, stimulates tumor cytolysis, expression of mRNA encoding TNF, and suppression of INF-gamma-induced expression of Ia Ag. Malondialdehyde-LDL fails to elicit these responses. We conclude that macrophage activation produced by maleyl-albumin is mediated by interaction with the low affinity, high capacity binding site for maleyl-albumin rather than the scavenger receptor.
Journal of Immunology, Sep 1, 2018
Journal of Immunology, Apr 15, 1987
Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-med... more Modulation of protein expression during interferon-gamma (IFN-gamma)-lipopolysaccharide (LPS)-mediated macrophage tumoricidal activation has been examined by metabolic radiolabeling of various murine peritoneal macrophage populations with [35S]methionine followed by SDS-PAGE analysis. Although both IFN-gamma and LPS are capable of stimulating the expression of several proteins when used independently, combined treatment induced the enhanced or de novo expression of a 120,000 dalton polypeptide. The expression of this protein was synergistically regulated by both IFN-gamma and LPS in a manner strongly reminiscent of the functional synergism that these two agents exhibit with respect to induction of tumoricidal activity. p120 expression could be seen first at approximately 3 hr after the addition of both agents, reached optimal expression by 6 hr, and maintained elevated synthesis for up to 24 hr. This time course corresponds closely to that seen for the acquisition of tumoricidal competence. Macrophages elicited in the primed state of activity in vivo with methyl vinyl ether co-polymer II (MVE-II) did not express p120, but could be induced to do so when treated with low doses of LPS. Under similar conditions, MVE-II-elicited cells also acquire tumoricidal activity. Macrophages obtained from mice chronically infected with bacillus Calmette-Guerin constitutively expressed both p120 and cytolytic activity. If such macrophages were cultured for 24 hr, the expression of both events decayed and was lost, but could be restored by treatment with low doses of LPS. Thus the data support a strong correlation between the expression by macrophages of a novel 120,000 dalton protein and the expression of tumor cytotoxicity.
Journal of Immunology, Aug 15, 1987
Maleyl-BSA and fucoidan induce expression of a set of early proteins in murine mononuclear phagoc... more Maleyl-BSA and fucoidan induce expression of a set of early proteins in murine mononuclear phagocytes.
Journal of Immunology, Sep 1, 1994
IFN-y and LPS have both been shown to stimulate enhanced chemoattractant cytokine gene expression... more IFN-y and LPS have both been shown to stimulate enhanced chemoattractant cytokine gene expression i n mononuclear phagocytes. In this report, IFN-y was found to suppress LPS-induced chemokine mRNA expression in a cell type-and gene-specific fashion. Expression of j E (monocyte chemoattractant protein-1) and KC (GRO/melanoma growth-stimulating activity) mRNA in macrophages stimulated with LPS was markedly suppressed by IFN-y in a dose-and time-dependent fashion. LPS-induced IP-10 mRNA was unaffected by IFN-y under identical experimental conditions. This effect was cell type-specific because ]E and KC mRNA expression in LPS-stimulated murine endothelial cells, TNF-a-stimulated endothelial cells, and NIH-3T3 cells were unaffected by IFN-y. The IFN-y-mediated suppression of LPS-stimulated KC mRNA expression was independent of protein synthesis and mediated at the transcriptional level. These observations indicate that IFN-y may function as a negative regulatory signal for the expression of some proinflammatory cytokines in macrophages. The cell type-dependent differential behavior of individual members of the chemokine family may be an important determinant of the cellular composition and outcome of an inflammatory response.
Journal of Immunology, Jun 15, 1997
Regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Localization of an intron EBV-... more Regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Localization of an intron EBV-responsive enhancer and characterization of its cognate GC-box binding factors.
PubMed, Oct 1, 1999
Interleukin 12 (IL-12) is known to play an important role in the development of an antitumor resp... more Interleukin 12 (IL-12) is known to play an important role in the development of an antitumor response. Its activity has been shown to be dependent upon the intermediate production of IFN-gamma and the influx into the tumor of CD8 lymphocytes. In a murine model, tumor regression induced by IL-12 treatment correlated with IFN-gamma, IP-10, and Mig expression in the tumor bed and was abrogated by antibodies to both chemokines. Here we examined the effects of rHuIL-12 on IFN-gamma and CXC chemokine gene expression in patients with renal cell carcinoma (RCC) in an attempt to determine whether a similar series of molecular events leading to IL-12-mediated tumor regression in mice is also detectable in humans. As in the murine RENCA model, cultured RCC cells themselves could be induced by IFN-gamma to synthesize IP-10 and Mig mRNA. Explanted RCC produced IFN-gamma and IP-10 mRNA in response to IL-12 treatment, which was consistent with the finding that biopsied RCC tumors from IL-12-treated patients also variably expressed augmented levels of those molecules after therapy. Although Mig mRNA was present in the majority of biopsied tumors prior to treatment, both the Mig and IP-10 chemokines as well as IFN-gamma were induced in the peripheral blood mononuclear cells of IL-12-treated patients. Skin biopsies of IL-12-treated patients also all synthesized IP-10 mRNA. This study demonstrates that recombinant human IL-12 therapy of patients with RCC has the potential to induce the expression of gene products within the tumor bed that may contribute to the development of a successful antitumor response.
Journal of Immunology, May 1, 1991
IFN-γ-Inducible Protein 10 (IP-10; CXCL10)-De cient Mice Reveal a Role for IP-10 in Effector T Ce... more IFN-γ-Inducible Protein 10 (IP-10; CXCL10)-De cient Mice Reveal a Role for IP-10 in Effector T Cell Generation and Tra cking J Immunol (April,2002) iPS cells but not their hematopoietic derivatives are immunogenic in the autologous recipient (P2210)
PubMed, Oct 15, 1994
Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (R... more Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (p50) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that RelA, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any RelA in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients may impair their capacity to respond normally to various stimuli.
Journal of Immunology, Jul 1, 1987
The surface expression of class II major histocompatibility molecules (immune associated or Ia an... more The surface expression of class II major histocompatibility molecules (immune associated or Ia antigens) is an acquired property of macrophages, essential to their ability to interact effectively with T lymphocytes. Surface expression of Ia is induced by stimulants such as interferon-gamma and is suppressed by agents such as lipopolysaccharide (LPS). Recent studies on several cultured cell lines indicate that interferon-gamma can heighten cellular levels of mRNA encoding Ia, and the level of such mRNA may represent an important regulatory focus for controlling expression of surface Ia. Murine peritoneal macrophages were treated with interferon-gamma and/or LPS and expression of Ia mRNA determined by Northern blot analysis with a probe specific for the murine beta-chain of I-A. mRNA specific for I-A beta was not detectable in explanted macrophages obtained from sites of sterile inflammation but was induced by treatment of purified recombinant interferon-gamma. This effect was dose dependent and was optimal by 24 hr after stimulation. Ia-specific mRNA preceded the surface expression of Ia as monitored by a radioimmunoassay using a monoclonal antibody specific for I-A beta. When a physiologic dose of LPS was added concomitantly with the interferon-gamma, the time course of induction if Ia-specific mRNA was not altered, but the amount of such mRNA detected was suppressed 40 to 80%. This effect was dependent on the dose of LPS, and the levels of mRNA correlated closely with subsequent surface expression of Ia. The ability of LPS to suppress both mRNA and cell surface Ia expression required that the suppressive agent be added within 12 hr of the inducing stimulus. This is the time frame during which accumulation of mRNA occurs. Thus the data demonstrates that accumulation of specific mRNA is a major regulatory focus governing expression of Ia both by interferon-gamma and LPS.
Journal of Immunology, Apr 1, 1989
Poxviral infection elicits human neutralizing antibodies recognizing diverse epitopes of the majo... more Poxviral infection elicits human neutralizing antibodies recognizing diverse epitopes of the major vaccinia virus surface protein D8 (VIR4P.1016)
Journal of Immunology, Sep 1, 1996
IL-4-induced STAT6 suppresses IFN-gamma-stimulated STAT1-dependent transcription in mouse macroph... more IL-4-induced STAT6 suppresses IFN-gamma-stimulated STAT1-dependent transcription in mouse macrophages.
Incontinence Transient receptor potential vanilloid 4 (TRPV4; VRL2) Studies in rodents suggest th... more Incontinence Transient receptor potential vanilloid 4 (TRPV4; VRL2) Studies in rodents suggest that antagonizing TRPV4 could help treat overactive bladder. In a mouse model of bladder dysfunction, Trpv4 deficiency decreased voiding frequency compared with that of wild-type controls. In mouse and rat models of bladder dysfunction, a selective TRPV4 antagonist decreased voiding frequency compared with vehicle control. Next steps include studying the molecular mechanisms that allow TRPV4 to sense the filling state of the bladder and performing medicinal chemistry optimization of the antagonist.
Journal of Immunology, Nov 15, 1986
The functional and biochemical responses of macrophages derived from the A/J mouse strain to IFN-... more The functional and biochemical responses of macrophages derived from the A/J mouse strain to IFN-gamma have been studied. As compared to macrophages obtained from C57BL/6 strain mice, cells from mice of the A/J strain are deficient in their response to IFN-gamma for acquisition of tumoricidal competence. This deficiency was not due to reduced expression of surface receptors for IFN-gamma or to altered affinity of the receptor for its ligand. IFN-gamma recently has been shown to enhance the potential activity of protein kinase C (PKc) and to modulate the efflux of intracellular Ca2+ in macrophages from C57BL/6 mice. Neither of these two biochemical changes were induced in macrophages derived from A/J mice. Functional competence could, however, be pharmacologically induced in both C57BL/6- and A/J-derived macrophages by combined treatment with an ionophore plus phorbol myristic acetate, which increase intracellular Ca2+ and stimulate PKc, respectively. Although the exact nature of the deficit in A/J strain mice has not been defined, the present findings indicate that it lies between the expression of receptor and the modulation of PKc activity and Ca2+ levels. Furthermore, the data provide support for the notion that these molecular changes are important components of the stimulus-response coupling process in IFN-gamma-mediated activation of macrophages.
PubMed, Feb 1, 2000
Purpose: The development of an effective antitumor immune response is compromised in patients wit... more Purpose: The development of an effective antitumor immune response is compromised in patients with renal cell carcinoma. Despite significant infiltration by T lymphocytes into renal tumors, no detectable induction of gene expression is associated with the generation of an antitumor immune response. Tumor-induced down-regulation of interleukin (IL)-2 expression may contribute to the impaired development of the T cell-mediated antitumor immune response. Within renal tumors, there is no detectable expression of IL-2 or the IL-2 receptor alpha chain, and only low levels of interferon gamma (IFN-gamma) mRNA are detected. Products in the tumor environment may suppress the expression of these genes, thus inhibiting production of type 1 helper T cell cytokines. Methods: Peripheral blood lymphocytes obtained from healthy volunteers were exposed to supernatants from renal cell carcinoma explants, and the immunologic consequences of this were assessed using a variety of molecular assays. Results: Soluble products from renal tumor explants can inhibit the production of IL-2 and IFN-gamma by peripheral blood lymphocytes and can suppress T-cell proliferation. Soluble products from renal cell carcinoma explants appear to block the nuclear translocation of nuclear factor kappa B (NFkappaB) proteins p50 and RelA without affecting cytoplasmic levels of these proteins. In some experiments, a reduction in the nuclear translocation of other transcription factors involved in IL-2 gene expression, including nuclear factor of activated T cells and accessory protein-1, was observed. Gangliosides isolated from tumor supernatants blocked the production of IL-2 and IFN-gamma in response to ionomycin plus phorbol myristate acetate stimulation. These gangliosides also inhibited stimulus-dependent activation and nuclear accumulation of NFkappaB. Coculture experiments demonstrated that renal cell carcinoma lines known to express gangliosides could inhibit the activation of NFkappaB in normal T cells and the Jurkat T-cell line. Supernatants from renal cell carcinoma explants and renal cell carcinoma cell lines can also suppress the proliferation of normal T cells, thus reproducing another defect observed in tumor-infiltrating lymphocytes. Supernatants from renal cell carcinoma tumors also appear to inhibit signaling through the IL-2 receptor. Although tumor supernatants had little effect on IL-2 receptor (alpha, beta or gamma) expression, they did block expression of JAK3, a key kinase involved in signaling through the IL-2 receptor pathway. Moreover, downstream events in IL-2 receptor signaling linked to JAK3 were impaired in T cells treated with tumor supernatants. Conclusion: These findings suggest that soluble products from renal tumors may suppress T-cell responses by blocking both IL-2 production and normal IL-2 receptor signaling.
Journal of Immunology, Apr 1, 1985
Macrophage activation in vivo has been associated with qualitative and quantitative alterations i... more Macrophage activation in vivo has been associated with qualitative and quantitative alterations in the release and metabolism of arachidonic acid. In the present study, we examined the effect of in vitro macrophage activation with recombinant gamma-interferon (IFN-gamma) on arachidonic acid secretion induced by exposure to a variety of stimulating agents. Secretion stimulated by challenge with unopsonized zymosan, insoluble immune complexes, the calcium ionophore A23187, or combinations thereof was unaltered in IFN-gamma-treated macrophages. However, when phorbol diesters active as tumor promoters were employed as challenge agents, arachidonate secretion was enhanced as much as 10-fold over that seen in nonactivated controls. The enhanced secretory response to PMA was detectable as early as 1 hr after exposure to IFN-gamma, reached a maximum within 3 to 6 hr, and subsequently declined to control levels even in the continued presence of the agent. Treatment with IFN-gamma did not alter the pattern of individual metabolites produced by macrophages challenged with either zymosan or PMA. Finally, the sensitivity to phorbol diesters was also increased by treatment with IFN-gamma (ED50 reduced from 35 ng/ml to 4 ng/ml). Thus, IFN-gamma could prime macrophages for a substantially amplified response to phorbol esters. Because the cellular mediator of PMA action has been identified as a Ca++, phospholipid-dependent protein kinase, a role for this enzyme in macrophage functional development is indicated.
Journal of Immunology, 1985
Synergistic action of A23187 and phorbol ester on human B cell activation.
Journal of Immunology, Sep 1, 1994
The mechanisms involved in negative regulation of IFN-gamma-induced IP-10 mRNA expression by IL-4... more The mechanisms involved in negative regulation of IFN-gamma-induced IP-10 mRNA expression by IL-4 have been examined in an immortalized murine macrophage cell line (ANA-1). As in primary peritoneal macrophages, IL-4 selectively inhibits the production of IP-10 mRNA by IFN-gamma-treated ANA-1 cells. Expression of another IFN-gamma-inducible gene (D3) was not affected by co-treatment with IL-4. Suppression of IFN-gamma-induced IP-10 mRNA expression by IL-4 is mediated at the level of transcription. IFN-gamma-induced transcription of CAT expression driven by a 243-bp IP-10 promoter fragment was also sensitive to the suppressive effects of IL-4. In contrast, LPS-induced CAT expression was unaffected under identical experimental conditions. The positive transcriptional response to IFN-gamma required an interferon stimulus response element (ISRE) sequence motif located approximately 230 bp upstream from the transcription start site of the IP-10 gene. That this site is the target of the suppressive action of IL-4 is indicated by the ability of IL-4 to inhibit IFN-gamma-mediated transcription from this ISRE sequence in the context of a heterologous promoter. Finally, both IFN-gamma and IL-4 can enhance or induce expression of distinct nuclear factors that exhibit ISRE-specific binding activity. IL-4 does not suppress the IFN-gamma-induced ISRE binding activity. Together, these findings demonstrate that IL-4 inhibits IP-10 mRNA production in murine macrophages by suppressing the formation of new transcripts. Both positive and negative transcriptional activity appears dependent on activation of factors that recognize the ISRE.
Journal of Immunology, Jun 1, 1986
Phorbol esters and dioctanoylglycerol block anti-IgM-stimulated phosphoinositide hydrolysis in th... more Phorbol esters and dioctanoylglycerol block anti-IgM-stimulated phosphoinositide hydrolysis in the murine B cell lymphoma WEHI-231.
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Papers by Thomas Hamilton