Heparan sulfate proteoglycans mediate interleukin-7-dependent B lymphopoiesis.

Heparin/heparan sulfate proteoglycans (HSPGs) have the potential to bind and directly regulate the bioactivity of hematopoietic growth factors including interleukin-7 (IL-7), a cytokine critical for murine B-cell development. We examined the consequence of manipulating soluble heparin and cell-surface heparan sulfate to IL-7-dependent responses of B-cell precursors. Soluble heparin was found to inhibit production of lymphoid, but not myeloid, cells in long-term bone marrow cultures. Analysis of pro-B cells lacking plasma membrane HS suggests that this glycosaminoglycan is required for efficient binding and responsiveness to IL-7. By contrast, responses of hematopoietic cells to other cytokines were not influenced by heparin addition or HS removal. Therefore, HSPGs on B-lineage precursors may function as IL-7 receptor components similar to HSPGs known to be important for the bFGF receptor. Other experiments suggest that HSPGs on the surface of stromal cells provide a weakly associating docking site for IL-7, possibly controlling availability of this cytokine to B-cell precursors. Together these data demonstrate a direct role for heparinlike molecules in regulating the IL-7-dependent stages of murine B lymphopoiesis.

Stromal cell modulation of negative regulatory signals that influence apoptosis and proliferation of B lineage lymphocytes.

The bone marrow microenvironment influences whether a given B cell proliferates, differentiates, or undergoes apoptosis. In this report, we demonstrate that apoptosis of primary murine B lymphocyte precursors can be regulated either positively or negatively by stroma. Several stromal lines that support lymphocyte outgrowth suppressed the spontaneous apoptosis of pre-B cells by as much as 90%. Direct contact with stromal cells more effectively protected lymphocytes than did stromal cell-CM or a collection of recombinant cytokines. In contrast, one unique stromal cell clone actually induced lymphocyte apoptosis, and a second line appeared inert. A survey of adherent cell lines suggested that expression of life-sparing molecules is widespread but not ubiquitous. Experiments with neutralizing Abs to CD44, vascular cell adhesion molecule-1 (VCAM-1), CD9, intercellular adhesion molecule-1 (ICAM-1), or ICAM-2 suggested that these interaction molecules do not deliver short-term survival signals to B cell precursors. Of particular interest, direct interaction with lymphocyte-supportive stromal cells minimized the negative regulatory effects of IL-1alpha, and a glucocorticoid, but not IFN-beta or PGE2. These results demonstrate that the effect of negative regulators depends upon the context in which these signals are presented. As molecules that influence B lymphopoiesis are better defined, it will be important to consider the role of each in combination with other stimuli.

Nonprotective effects of extracellular metallothionein.

Metallothionein (MT) is a small, cysteine-rich protein that is readily induced by exposure to heavy metal cations. In previous work, we have demonstrated that MT has several significant immunomodulatory properties. MT decreases antigen-specific humoral responses in vivo and inhibits the ability of T cells to proliferate in response to antigen presented in vitro. To further characterize the mechanism by which this protein inhibits responsiveness to antigen, we have examined the effects of MT on cell viability in an antigen-presentation assay. MT (20 microM) caused substantial death to both lymphocytes and monocytes after 3 days of culture. The observed toxicity cannot be attributed to either increased superoxide radical generation or to production of tumor necrosis factor by MT-treated macrophages. Fractionation of supernatants from MT-treated cells suggests that the agent responsible for causing cytotoxicity is a soluble factor of at least 30 kDa. These results counter the perception that metallothionein uniformly plays a protective role in metal-stressed individuals.

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.

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.

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.