Michael A. Lynes

Immunomodulatory activities of extracellular metallothionein. I: Metallothionein effects on antibody production

Extracellular metallothionein (Zn,Cd-MT) has previously been shown to be a potent inducer of lymphocyte proliferation and to synergize with polyclonal activators in proliferation assays. In this report, the effects of metallothionein on the development of humoral responsiveness are examined. In vivo, the specific anti-ovalbumin (OVA) IgG response was diminished by co-injection of Zn, Cd-MT, while total IgG levels remained unchanged. A similar reduction was also observed when Zn,Cd-MT was administered during the development of an anti-sheep red blood cell (sRBC) humoral response. When amounts of Zn and Cd equimolar to that associated with the Zn, Cd-MT were co-injected with OVA, humoral responsiveness was enhanced, in contrast to the suppression seen with Zn, Cd-MT. Apothionein lacking the available thiols associated with native Zn, Cd-MT had no effect on the development of humoral immunity. These results point to the thiols associated with the protein as the important determinants in the observed immunosuppression and this is supported by the capacity of UC1MT, a new monoclonal anti-MT antibody, to reverse MT mediated immunosuppression. No evidence was found to suggest that Zn,Cd-MT was interacting directly with OVA. Finally, in vitro experiments with LPS-stimulated splenocyte production of IgM correlated with the in vivo observations of Zn,Cd-MT. These data provide evidence for a significant role for MT in the development of metal-mediated immunomodulation and suggest that MT may also possess immunomodulatory functions under circumstances where MT is synthesized in the absence of heavy metal stress. Furthermore, it may be possible to take advantage of this system to exogenously manipulate the development of the immune response.

Interactions of metallothionein with murine lymphocytes: plasma membrane binding and proliferation.

Metallothionein (MT) is a thiol rich protein that has been well characterized for its ability to bind and sequester heavy metal cations, free radicals and other reactive toxicants. In addition to induction by these stressors, MT gene expression is upregulated by several cytokines of the acute phase response. In previous work, we have shown that MT can alter aspects of lymphocyte function. MT alone induces modest proliferation of unfractionated splenocytes and acts synergistically with T cell- and B cell-specific mitogens. In contrast, MT inhibits humoral responsiveness in vivo and reduces in vitro T cell responses to processed antigen. In this report, we describe the effects of MT on specific lymphocyte subpopulations in order to further characterize the mechanism of MT-mediated alterations of immune activity. MT binds to the plasma membrane of both T and B lymphocytes, but, in the absence of a costimulatory agent, MT induces lymphoproliferation only in B cells. MT also enhances the capacity of naive B lymphocytes to differentiate into plasma cells. These results demonstrate differential immunomodulatory activities of MT and may explain some of the diverse immunoregulatory effects associated with exposure to environmental toxins.

Immunomodulatory activities of extracellular metallothionein. II. effects on macrophage functions

Metallothionein (MT) is a thiol-rich protein that is rapidly induced by exposure to heavy metal cations. We have previously demonstrated that exogenous MT stimulates murine splenocytes to proliferate, but inhibits humoral responses to antigen. These observations suggest that metallothionein released from cells has a complex role in heavy metal-mediated immune dysfunction. Here we examine one possible mechanism by which MT mediates suppression of humoral immunity. Exposure of macrophages to 20 microM MT did not affect their ability to engulf opsonized sheep erythrocytes, but in the presence of 20 microM MT, peritoneal macrophages were stimulated to produce increased levels of oxygen radicals. These results correlated with observations that while macrophage phagocytosis of opsonized Candida albicans was unaltered by the presence of exogenous MT, killing of the engulfed yeast cells was dramatically enhanced by 20 microM MT. Amounts of free cadmium and zinc equimolar to that added as Zn,Cd-MT had no effect on candidacidal activity. MT was also found to significantly decrease lymphocyte proliferation mediated by macrophage activity. Biotinylated MT (MT-b) bound specifically to the plasma membranes of these macrophages, suggesting that membrane-associated molecules of the macrophage may transduce a signal mediated by MT binding. These results demonstrate that macrophages are a sensitive target for MT-mediated immunomodulation and that some of the consequences of the MT interaction with macrophages may be alterations in the capacity to produce an effective immune response and increased extracellular exposure to damaging free radicals.

The physiological roles of extracellular metallothionein.

Metallothionein (MT) is a low-molecular-weight protein with a number of roles to play in cellular homeostasis. MT is synthesized as a consequence of a variety of cellular stressors, and has been found in both intracellular compartments and in extracellular spaces. The intracellular pool of this cysteine-rich protein can act as a reservoir of essential heavy metals, as a scavenger of reactive oxygen and nitrogen species, as an antagonist of toxic metals and organic molecules, and as a regulator of transcription factor activity. The presence of MT outside of cells due to the Influence of stressors suggests that this protein may make important contributions as a “danger signal” that influences the management of responses to cellular damage. While conventional wisdom has held that extracellular MT is the result of cell death or leakage from stressed cells, there are numerous examples of selective release of proteins by nontraditional mechanisms, including stress response proteins. This suggests that MT may similarly be selectively released, and that the pool of extracellular MT represents an important regulator of various cellular functions. For example, extracellular MT has effects both on the severity of autoimmune disease, and on the development of adaptive immune functions. Extracellular MT may operate as a chemotactic factor that governs the trafficking of inflammatory cells that move to resolve damaged tissues, as a counter to extracellular oxidant-mediated damage, and as a signal that influences the functional behavior of wounded cells. A thorough understanding of the mechanisms of MT release from cells, the conditions under which MT is released to the extracellular environment, and the ways in which MT Interacts with sensitive cells may both illuminate our understanding of an important control mechanism that operates in stressful conditions, and should indicate new opportunities for therapeutic management via the manipulation of this pool of extracellular MT.

Stress proteins as agents of immunological change: some lessons from metallothionein

The stress response proteins each have somewhat unique characteristics that enable them to function under conditions of cellular stress, and to contribute to cellular survival in difficult times. The immune response is, by definition, a mechanism that often operates in times of cellular stress, and even creates stress during its operation. Cells called upon to respond to tissue damage caused by inflammation can have extraordinary demands placed upon them and surrounding tissue may suffer damaging conditions that were originally established to eliminate the source of the inflammation. Stress proteins may be released from some of these damaged cells as a programmed response to the stress, or as a simple consequence of excessive damage to the plasma membrane. In either instance, there is the opportunity for these stress proteins to interact with cells and proteins in the extracellular environment. It may be that those same characteristics that enable stress proteins to interact with structures within the cell also enable interactions outside the cell, but with dramatically different results. As has been found with MT, interference with these extracellular interactions may decrease the consequences of stress on the immune response, and may enable more effective immunity. It may also be possible to employ the various stress proteins to manipulate normal immune function.

Heavy metal ions in normal physiology, toxic stress, and cytoprotection

Abstract:  As a group, heavy metals include both those essential for normal biological functioning (e.g., Cu and Zn), and nonessential metals (e.g., Cd, Hg, and Pb). Both essential and nonessential metals can be present at concentrations that disturb normal biological functions, and which evoke cellular stress responses. The cellular targets for metal toxicity include tissues of the kidney, liver, heart, and the immune response and nervous systems. Intriguingly, manipulations of specific metals, their reservoirs, and the cellular stress response can have therapeutic effects on certain diseases. In this minireview, we will consider both the biological responses to stressful levels of heavy metal cations, and experimental and clinical manipulations of these cations as a means to improve human health parameters.

A Microarray Biosensor for Multiplexed Detection of Microbes Using Grating-Coupled Surface Plasmon Resonance Imaging

Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a nontoxic form of Pseudomonas aeruginosa exotoxin A (ntPE), Bacillus globigii, Mycoplasma hyopneumoniae, Listeria monocytogenes, Escherichia coli, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free, and highly multiplexed assay requiring nanoliter amounts of capture reagents.

Antigen-specific T cell phenotyping microarrays using grating coupled surface plasmon resonance imaging and surface plasmon coupled emission.

The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individuals medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individuals response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities were used to detect and characterize CD4(+) T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabeled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.

Mechanisms of anemia in SHP-1 protein tyrosine phosphatase-deficient "viable motheaten" mice

OBJECTIVE Viable motheaten mice (abbreviated gene symbol me(v)) are deficient in SHP-1, a critical negative regulator of signal transduction in hematopoietic cells. These mice exhibit severe immune dysfunction accompanied by hyperproliferation of myeloid cells, widespread inflammatory lesions, and regenerative anemia. The aim of this study was to investigate the mechanisms underlying anemia in me(v)/me(v) mice. MATERIALS AND METHODS Multiple hematologic parameters, osmotic fragility, and erythropoietin levels were measured to characterize the anemia in me(v)/me(v) mice. B-cell-deficient me(v)/me(v) Igh-6(null) mice were generated to assess the role of anti-erythrocyte antibodies. Coombs assays and flow cytometry were carried out for detection of anti-erythrocyte antibodies. Oxidant production by macrophages, glutathione levels, and lipid peroxidation products in erythrocytes were measured, as was the impact of oxidant on the ultrastructure of me(v)/me(v) erythrocytes. Erythroid maturation and erythrocyte plasma membrane integrity were assessed with flow cytometry by evaluating CD71 expression and annexin V labeling. RESULTS The regenerative anemia of me(v)/me(v) mice was associated with erythrocyte changes that were independent of the presence of anti-erythrocyte antibodies. Erythrocytes from me(v)/me(v) mice had increased fragility and heightened susceptibility to oxidant damage. Macrophages from me(v)/me(v) mice demonstrated a higher basal level of oxidant production and enhanced production after stimulation. Oxidant damage in me(v)/me(v) erythrocytes was evidenced by a significant elevation of lipid peroxidation and diminished levels of glutathione. CONCLUSION Our results support the hypothesis that as a consequence of severe inflammatory disease, me(v)/me(v) erythrocytes are subject to exceptionally high oxidative stress resulting in oxidation of phospholipids in the erythrocyte membrane with subsequent hemolysis.

Biomonitoring in the Era of the Exposome.

Background: The term “exposome” was coined in 2005 to underscore the importance of the environment to human health and to bring research efforts in line with those on the human genome. The ability to characterize environmental exposures through biomonitoring is key to exposome research efforts. Objectives: Our objectives were to describe why traditional and nontraditional (exposomic) biomonitoring are both critical in studies aiming to capture the exposome and to make recommendations on how to transition exposure research toward exposomic approaches. We describe the biomonitoring needs of exposome research and approaches and recommendations that will help fill the gaps in the current science. Discussion: Traditional and exposomic biomonitoring approaches have key advantages and disadvantages for assessing exposure. Exposomic approaches differ from traditional biomonitoring methods in that they can include all exposures of potential health significance, whether from endogenous or exogenous sources. Issues of sample availability and quality, identification of unknown analytes, capture of nonpersistent chemicals, integration of methods, and statistical assessment of increasingly complex data sets remain challenges that must continue to be addressed. Conclusions: To understand the complexity of exposures faced throughout the lifespan, both traditional and nontraditional biomonitoring methods should be used. Through hybrid approaches and the integration of emerging techniques, biomonitoring strategies can be maximized in research to define the exposome. Citation: Dennis KK, Marder E, Balshaw DM, Cui Y, Lynes MA, Patti GJ, Rappaport SM, Shaughnessy DT, Vrijheid M, Barr DB. 2017. Biomonitoring in the era of the exposome. Environ Health Perspect 125:502–510; http://dx.doi.org/10.1289/EHP474