Protein tyrosine kinase activation by polycyclic aromatic hydrocarbons in human HPB-ALL T cells.

It has been well established that certain polycyclic aromatic hydrocarbons (PAHs), such as 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (3MC), and benzo[a]pyrene (BaP), produce immunotoxicity and cancer in rodents and that these effects are also likely seen in humans. Our laboratory has found that polycyclic aromatic hydrocarbons (PAHs) produce an increase in intracellular Ca2+ in lymphocytes that appears to correlate with their immunotoxicity. Specifically, immunotoxic PAHs, such as DMBA and BaP, have been shown to produce a sustained increase in intracellular Ca2+ in lymphocytes, whereas nonimmunosuppressive PAHs, such as benzo[e]pyrene (BeP) and anthracene, do not. Our studies previously demonstrated that the rapid increase in intracellular Ca2+ produced by DMBA in HPB-ALL T cells was caused by protein tyrosine kinase (PTK) activation in human T cells, leading to tyrosine phosphorylation of phospholipase C (PLCgamma) and IP3-dependent Ca2+ mobilization. However, the specificity of PTK activation by PAHs was not established. In the present studies, we extend our observations of PTK activation by examining a number of PAHs for their effects on total and specific (Fyn and ZAP-70) PTK activity. We show that 10 microM concentrations of PAHs nonspecifically and rapidly (within 5 min) stimulate PTKs in the HPB-ALL human T cell line. BeP and anthracene were found to be nearly as effective at increasing total tyrosine kinase activity as DMBA, 3MC, and BaP, observed 5 min after exposure. We found that only immunotoxic PAHs activated the Fyn and ZAP-70 PTKs at 10 min, but total PTK activity was still increased by nonimmunotoxic PAHs, BeP, or anthracene after 10 min of exposure. These studies demonstrate that immunotoxic PAHs increase total and specific PTK activity in the human HPB-ALL T cell line. Thus the rapid increase in PTK activity produced by PAHs may not correlate with the immunotoxicity of these agents.

Characterization of intracellular calcium responses produced by polycyclic aromatic hydrocarbons in surface marker-defined human peripheral blood mononuclear cells.

Previous studies have demonstrated that polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP) and 7,12-dimethybenz[a]anthracene (DMBA), and possibly 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), may exert their immunosuppressive effects by altering intracellular Ca2+ homeostasis in lymphocytes. In these studies, we examined the effects of two immunosuppressive PAHs (BaP and DMBA), two nonimmunosuppressive PAHs (benzo[e]pyrene (BeP) and anthracene (ANTH)), and TCDD on intracellular Ca2+ levels in surface marker-defined human peripheral blood mononuclear cells (HPBMC). BaP and DMBA, but not BeP and ANTH, were found to produce a time-dependent increase in intracellular Ca2+ with maximal effects achieved following 42- to 66-hr exposures. In a series of studies with HPBMC obtained from 10 donors exposed in vitro for 42 hr, BaP and DMBA were found to produce a significant increase in Ca2+ in CD3+ T cells, CD19+ B cells, and CD14+ monocytes. BeP and ANTH did not produce a statistically significant increase in Ca2+ in the group of donors, but occasionally produced an apparent nonspecific elevation of Ca2+ in HPBMC from individual donors. Interestingly, TCDD produced a small and statistically significant increase in Ca2+ only in B cells analyzed for the pooled 10 donors. Certain BaP metabolites, such as the 7,8-dihydrodiol and the 7,8-diol-9,10-epoxide, were more effective in elevating Ca2+ in HPBMC lymphocytes at 20 hr than was BaP. These results demonstrate in normal HPBMC that immunosuppressive PAHs alter intracellular Ca2+ homeostasis in B cells, T cells, and monocytes, and suggest that P450 metabolism may play an important role in the immunotoxicity of certain PAHs.

Human T cells are highly sensitive to suppression of mitogenesis by polycyclic aromatic hydrocarbons and this effect is differentially reversed by alpha-naphthoflavone.

The immunosuppressive effects of polycyclic aromatic hydrocarbons (PAHs) on immune responses in rodents, both in vivo and in vitro, have been widely documented. However, few studies have addressed the immunotoxicity of PAHs in the human system. In this report, we examined the toxic effects of nine different PAHs on human peripheral blood T cell mitogenesis. We found that benzo(a)pyrene (BaP), 3-methylcholanthrene (3-MC), and 7,12-dimethylbenz(a)anthracene (DMBA) were highly immunotoxic in the human system, while dibenz(a,c)anthracene (DAC) and dibenz(a,h)anthracene (DAH) were of intermediate toxicity, 9,10-dimethylanthracene (DMA), benzo(e)pyrene (BeP), and benz(a)anthracene (BA) were mildly immunotoxic, and anthracene (ANTH) had no measureable toxicity at the concentrations tested. Our results using human lymphocytes differed from previous studies in rodents, in that BaP and 3-MC were the most immunotoxic PAHs in the human mitogenesis assay, while DMBA has long been regarded as the PAH that is most potently toxic to rodent T cell responses. We also showed that alpha-naphthoflavone (ANF), which functions as both an Ah receptor antagonist and an inhibitor of cytochrome P450 activity, was able to block the suppressive effects of both BaP and DMBA, but not 3-MC. This suggests that the immunotoxicity of 3-MC may be mediated through a different mechanism than BaP or DMBA. Addition of four different BaP metabolites directly to cultures of human mononuclear cells showed that the 7,8-dihydrodiol metabolite was the most toxic, and that this toxicity could be completely blocked by equimolar and 10-fold greater concentrations of ANF. The 7,8-dihydrodiol metabolite was probably further metabolized to the 7,8-diol epoxide, the toxicity of which could not be effectively reversed by ANF. The 4,5-epoxide metabolite was apparently cytotoxic at high concentrations (10 microM), while the 7-hydroxy metabolite had no overtly negative effects on proliferation.

Molecular mechanisms of toxicant-induced immunosuppression: role of second messengers.

Changes in immunocompetence following chemical exposure have been established for a wide variety of unrelated agents. For the vast majority of immunotoxic compounds thus far identified, disruption of normal immune function is clearly mediated through direct interactions between the agent, or its metabolite, and immunocompetent cells. Regardless of whether this interaction occurs at the level of the cell membrane or at intracellular sites, basic regulatory processes mediated by second messengers are often altered. These alterations can ultimately result in immunologic dysfunction, which is most often manifested as immunosuppression. The specific disruptions in intracellular signaling produced by a number of immunotoxic compounds have now been identified, leading to a basic understanding of their molecular mechanism of action. Equally important, through the application of these agents as biological probes, new insights have been gained pertaining to which intracellular processes control which cellular functions within various populations of immunocompetent cells.

Inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic hydrocarbons in HPB-ALL human T cells and other tissues.

Polycyclic aromatic hydrocarbons (PAHs) are immunosuppressive chemicals found in the environment that have been shown to disrupt intracellular Ca2+ homeostasis and Ca(2+)-dependent signaling in human and murine lymphocytes. Many PAHs produce a rapid and sustained increase in intracellular free Ca2+ in lymphocytes. The mechanism of persistent Ca2+ perturbation remains undefined. In the present studies, ATP-dependent 44Ca2+ uptake into vesicles prepared from a 15,000g supernatant of HPB-ALL human T cell lysates was significantly inhibited by 0.1, 1, and 10 microM concentrations of the immunotoxic PAHs 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BAP), benz[a]anthracene, and 9,10-dimethylanthracene, but not by the less immunotoxic compounds anthracene (ANT) and benzo[e]pyrene (BEP). Ca(2+)-ATPase catalytic activity was determined by quantitating hydrolysis of ATP in the presence or absence of PAHs, with known ATPase inhibitors included as controls. Formation of inorganic phosphate was significantly decreased (> 65% of control at 10 microM) by DMBA and BAP, whereas ANT and BEP caused only a slight reduction in activity (10% of control at 10 microM). Anthracene partially reversed the inhibitory effect of DMBA and BAP on ATP hydrolysis when agents were coincubated. Both DMBA and BAP, but not ANT and BEP, inhibited the activity of all known SERCA-type Ca(2+)-ATPases, while not affecting either Na+, K(+)-ATPase activity or plasma membrane Ca(2+)-ATPase activities. These results demonstrate that immunotoxic and carcinogenic polycyclic aromatic hydrocarbons have a thapsigargin-like effect in human lymphocytes and SERCA-containing tissues from various species. Inhibition of SERCA activity may play an important role in altered Ca2+ homeostasis in lymphocytes and other tissues.

Role of alterations in Ca(2+)-associated signaling pathways in the immunotoxicity of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are an important class of environmental pollutants that are known to be carcinogenic and immunotoxic. The effects of PAHs on the immune system of various animals and models have been studied for at least 30 yr. Despite these efforts, the mechanism or mechanisms by which PAHs exert their effects on the immune system are still largely unknown. During recent years, the molecular events associated with lymphocyte activation and receptor-mediated signaling have become increasingly clear. Substantial progress has been made in understanding the molecular and cellular bases for toxicant-induced immune cell injury. Understanding mechanisms of drug or chemical effects on the immune system is an important area of research in the field of immunotoxicology, and indeed in all fields of toxicology. Mechanistic toxicology plays an important role in risk assessment and extrapolation of potential human health effects. In this review, we have summarized recent evidence that has examined the effects of PAHs on the immune system of animals and humans. In particular, we have focused on the effects of PAHs on cell signaling in lymphoid cells and have examined the hypothesis that PAHs alter lymphocyte activation via calcium-dependent mechanisms. Previously published reports are discussed, and new data obtained with murine B cells and cell lines are presented demonstrating the relationship between alterations in intracellular calcium and immune dysregulation. These data demonstrate a strong association between PAH-induced alterations in B- and T-lymphocyte activation and changes in calcium homeostasis.

The comparative pulmonary toxicity of beryllium metal and beryllium oxide in cynomolgus monkeys.

Inhalation of beryllium (Be) may result in an immune-mediated, chronic granulomatous pulmonary disorder known as chronic beryllium disease (CBD). The physicochemical form of Be may affect the incidence and severity of CBD. We exposed cynomolgus monkeys, by bronchoscopic, intrabronchiolar instillation, to either beryllium oxide (BeO; heat-treated at 500 degrees C) or Be metal at concentrations selected to achieve equimolar concentrations of available Be2+ ions dissolving from the particles. Monkeys underwent bronchoalveolar lavage of the right and left diaphragmatic lobes at 14, 30, 60, 90, and 120 days post exposure (dpe). Monkeys were sacrificed at 80 and 180 dpe for evaluation of histopathological pulmonary changes. Numbers of lymphocytes from lung lobes of Be metal-exposed, but not BeO-exposed, monkeys were increased at 14, 30 and 90 dpe. Lung lymphocytes were increased for BeO exposed monkeys only at 60 dpe. In vitro, Be-specific, lung lymphocyte proliferation occurred at 14, 60, and 90 dpe for lymphocytes from Be metal-exposed lung lobes only. At no time were values from BeO-exposed lung lobes different from values from control lobes. Lung lesions in Be metal-exposed monkeys were characterized by focally intense, interstitial fibrosis, marked Type II cell hyperplasia, and variable lymphocyte infiltration. Some Be-metal-exposed monkeys had discrete immune granulomas consisting of tightly organized lymphocytic cuffs surrounding nodular aggregates of epithelioid macrophages. Lesions were rarely present in BeO-exposed monkeys and were much less severe. These data suggest that Be metal produces more severe pulmonary lesions than does BeO and that these lesions are accompanied by Be-specific immune responses.

The effects of age on immune responses in the antigen-instilled dog lung. Antibody responses in the lung and lymphoid tissues following primary and secondary antigen instillation.

To evaluate the effects of age on immunity induced by lung immunization, 11 aged (12-17 years; median age = 14) and 12 young (2-5 years) male Beagle dogs were instilled with 10 mg of keyhole limpet hemocyanin (KLH) in the right cardiac lung lobe and 10(10) sheep red blood cells (SRBC) in the left cardiac lung lobe. Five aged and six young dogs were sacrificed at day 9 after primary antigen instillation. The remainder were given challenge antigen instillations of KLH and SRBC at day 21 and sacrificed 7 days later. Serum, bronchoalveolar lavage fluid and lung tissue from immunized and control lobes, tracheobronchial, mesenteric and popliteal lymph nodes, spleen, and blood were taken at sacrifice. Anti-KLH IgA, IgG and IgM antibody production by cells in lung tissue and lavage fluid from the KLH-exposed lobe was lower at primary immunization and challenge in aged than young dogs. Lavage fluid IgA and IgG levels from the KLH exposed lobe at primary immunization and challenge were lower in aged versus young dogs, while IgM levels were lower only after primary immunization. Localized lung immune memory responses were also markedly lower in aged dogs when compared with young dogs. Anti-SRBC responses were similar to the anti-KLH responses. Our data show that systemic immune responses are significantly lower in aged dogs following primary antigen instillation, but not after antigen challenge in the lung. This was not the case for localized lung immune responses, which were significantly lower in aged dogs even following antigen challenge. The data also show that antibody production by lavage cells is a good index of interstitial lung cell antibody production.

Long-term consequences of 239PuO2 exposure in dogs: persistent T lymphocyte dysfunction.

Young Beagle dogs were exposed by inhalation to aerosols of 239PuO2 and observed for their lifespans as part of a large, ongoing study of the biological effects of inhaled radionuclides. The purpose of our study was to compare certain immune responses of the 239PuO2-exposed dogs at middle age (7-10 years old) and old age (12-14 years old), with those of unexposed, age-matched or young (3-4 years old) animals. Some of the aged, exposed dogs had developed lung tumours. Lymphocyte proliferative responses to phytohaemagglutinin (PHA) were lower in aged control dogs than in either young or middle-aged control dogs. Both aged and middle-aged, radiation-exposed dogs had decreased responses to PHA when compared to age-matched controls. Responses to concanavalin A (Con A) were not affected by age in control dogs, but tended to decrease in the oldest group of radiation-exposed dogs. Responses to both PHA and Con A were severely depressed in tumour-bearing dogs. The cytolytic activity of natural killer cells was not affected by age, radiation exposure, or tumour presence. We concluded that inhalation of 239PuO2 by young Beagle dogs resulted in an earlier-than-normal decrease in the ability of T cells to respond to mitogenic stimulation. In other words the depressed responses to PHA that were observed might represent radiation-induced, accelerated ageing of the T cell response.

Interferon-gamma-induced priming for secretion of superoxide anion and tumor necrosis factor-alpha declines in macrophages from aged rats.

Macrophage responses to recombinant IFN-gamma decline during aging, as measured by two criteria of macrophage activation, O2- and TNF-alpha secretion. The production of O2- by macrophages in response to opsonized-zymosan and recombinant rat IFN-gamma is 75% lower in 23-month-old rats than in 3-month-old rats. Furthermore, the secretion of TNF-alpha in response to IFN-gamma and LPS is almost absent in macrophages from aged rats. Production of both O2- and TNF-alpha by resident peritoneal macrophages from specific pathogen-free aged rats in response to priming and triggering stimuli was partially or fully restored by implantation of syngeneic pituitary grafts from young rats. These data demonstrate that macrophages from aged rats are defective in their response to a priming signal induced by IFN-gamma, and they suggest that impaired macrophage responses during aging may be reversible.

Sex differences in lectin-induced interleukin-2 synthesis in aging rats.

Several aspects of T cell-mediated immune responses decline with age, but it is not known how gender affects this decline. Using 3- and 26-month-old male and female Fischer 344 rats, we examined the effects of sex and age on four different immune events that normally decline during aging: antibody synthesis to a T-dependent antigen, lectin-induced proliferative responses, IL-2 synthesis, and natural killer activity. We found that all these responses decreased with age. Spleen cells from aged females had higher spontaneous, phytohemagglutinin (PHA), and concanavalin A (Con A)-induced proliferative responses, and a two-fold increase in IL-2 synthesis than aged males, although no differences in these responses were evident between young males and females. Both natural killer (NK) activity and the ability to generate plaque-forming cells to sheep erythrocytes (SRBC) declined with age, but there were no differences between males and females for these responses in either age group. These data indicate that sex-associated differences in IL-2 synthesis and spontaneous and lectin-induced proliferative responses that are not detected in young animals become evident with advancing age.

A pituitary-thymus connection during aging.

Thymic involution is a normal consequence of aging. It has often been speculated that if this age-associated atrophy of the thymus gland could be prevented, the natural decline that occurs in a number of T-cell-mediated immune responses could be reversed. It has recently become clear that thymic involution can indeed be reversed by altering the hormonal environment of aged animals. These data support the concept of an active and functional pituitary-thymus axis. Since thymic reconstitution can result in restoration of some T-cell responses, it would appear that intrinsic defects which exist in T cells of aged animals can be at least partially reversed. This suggests that the aged environment plays a greater role in the decline of T-cell functions than has been previously recognized. Furthermore, phorbol esters and calcium ionophores can restore suppressed proliferative responses of T cells from aged rodents, so we speculate that intrinsic defects in T cells of aged subjects lie between the recognition system for antigen/lectin and intracellular transmission of this signal.

A soluble 61-kDa protein is associated with inhibition of lectin-induced proliferation and IL-2 synthesis.

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH4(2)SO4 (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 micrograms of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, proteinaceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.

Chicken serum inhibits lectin-induced proliferation of autologous splenic mononuclear cells.

Splenic mononuclear cells (SMC) proliferate well in response to phytomitogens in serum-free medium. Low concentrations of corticosterone significantly inhibit both spontaneous (1 ng/ml) and phytohemagglutinin-induced proliferation (12 ng/ml) of chicken SMC. Addition of as little as 3% normal heat-inactivated chicken serum to both autologous and heterologous suspensions of chicken SMC causes a 50% inhibition in lectin-induced proliferation. This suppression by normal chicken serum increases in a dose-dependent manner. Inhibition of DNA synthesis by chicken serum is not due to endogenous corticosterone because charcoal stripping of the serum to deplete glucocorticoids does not remove the immunosuppression. Chicken serum is not cytotoxic to lymphoid cells. These data demonstrate that low, physiologic concentrations of corticosterone inhibit proliferation of chicken SMC. Utilization of a serum-free culture system also permitted the identification of a serum factor that appears to be even more immunosuppressive than corticosterone. Since this factor inhibits the lectin-induced proliferation of both autologous and heterologous avian lymphoid cells, it may be a very important naturally occurring immunoregulatory compound.

Role of growth hormone in regulating T-dependent immune events in aged, nude, and transgenic rodents.

Growth hormone (GH) appears to play a major role in a reciprocal axis that has been postulated between the thymus and pituitary glands. Our previous studies showed that thymic structure, as well as T-cell proliferation and IL-2 synthesis, could be restored in aged female Wistar-Furth rats by the implantation of GH3 pituitary adenoma cells. These cells secrete GH and some prolactin. We have now used three different approaches to determine whether GH affects a variety of immune events in vivo in both old and young rodents, and whether GH3 cells can directly affect progenitor T-cells in nude rats that congenitally lack a thymus gland. To test the effects of GH in aged rats, 750 micrograms of pituitary-derived ovine GH was injected 2 x daily into 26-month-old Fischer 344 rats for 5 weeks. This approach demonstrated that GH augments splenocyte proliferation to T-cell lectins as well as natural killer (NK) activity at low effector:target ratios even though morphologic characteristics of the thymus were not altered. To assess the effect of GH in young rodents, mice were studied that were transgenic for the rat metallothionein-GH gene. Histologic evaluation of thymus glands revealed that the amount of adipose tissue and the number of epithelial cells and Hassall's corpuscles are augmented in transgenic mice. Splenocyte proliferation at suboptimal mitogen doses is greater in transgenic than in control littermate mice, but neither IL-2 synthesis nor antibody synthesis to sheep erythrocytes is affected. The role of pituitary hormones on progenitor T-cells was then explored by implanting GH3 cells into Rowett nude rats.(ABSTRACT TRUNCATED AT 250 WORDS)