Qualitative and quantitative experimental models to aid in risk assessment for immunotoxicology.

We have previously reported on the design and content of a screening battery using a "tier" approach for detecting potential immunosuppressive compounds in mice [1]. This battery was composed of various immune function, immunopathology and host resistance tests, the results of which could help establish the potential of chemical and biological agents to cause immunosuppression. The data from these studies, which now encompass over 50 compounds, have been analyzed in an attempt to improve future testing strategies and provide information to aid in the risk assessment process. Specifically, the following two issues will be addressed; what are the likelihood(s) for each of the individual tests and testing configurations to accurately identify immunotoxic compounds? and what are the quantitative and qualitative relationships between the immune tests and host resistance assays?

Expansions of T-cell subsets expressing particular T-cell receptor variable regions in chronic beryllium disease.

Chronic beryllium disease (CBD) is a granulomatous disorder characterized by the presence of noncaseating granulomas and mononuclear cell inflammation, occurring in 1 to 5% of people exposed to beryllium in the workplace. In the lungs of affected patients, CD4(+) T cells accumulate. Using anti-T-cell receptor (TCR) monoclonal antibodies, we investigated the TCR beta and alpha variable (Vbeta and Valpha, respectively) repertoire in the bronchoalveolar lavage (BAL) and blood of both CBD patients and healthy controls. There was marked heterogeneity within the BAL CD4(+) T-cell repertoire in both patients and controls. However, 11 of the 28 CBD patients demonstrated 16 different T-cell subset expansions within the BAL as compared with only one expansion in ten healthy controls. Five of the 16 expansions in CBD patients expressed Vbeta3. Altered TCR expression within the BAL T-cell repertoire appeared to persist over time in patients who underwent repeat evaluation. After in vitro stimulation of BAL T cells with beryllium sulfate and interleukin-2, we noted further alteration of the BAL TCR repertoire in some individuals. These results provide additional insight into the involvement of CD4(+) T cells in this disease and form the basis for studies to examine the nature of the stimulating antigen.

Properties of rat tracheal epithelial cells separated based on expression of cell surface alpha-galactosyl end groups.

We used Griffonia (bandeiraea) simplicifolia I (GS I) lectin and flow cytometry to isolate subsets of rat tracheal epithelial cells based on the presence or absence of cell surface alpha-galactosyl end groups. These fractions were designated GS I-positive and -negative, respectively. Ninety-eight percent of the cells in the GS I-positive fraction expressed cell surface alpha-galactosyl end groups; 95% had immunocytochemically detectable keratin 14-related protein (a basal cell marker) and 98% lacked alcian blue-periodic acid-Schiff (AB-PAS)-stained cytoplasmic granules. More than 90% of the GS I-positive cells had a high nuclear-to-cytoplasm ratio, had tonofilaments, and lacked organelles characteristic of other differentiated cell types; they were thus classified as basal cells. In bioassays, the GS I-positive fraction had a colony-forming efficiency greater than or equal to that of native tracheal cell suspensions, and the cells were able to repopulate denuded tracheal grafts with ciliated, secretory, and basal cells. More than 99% of the cells in the GS I-negative fraction lacked cell surface alpha-galactosyl end groups, 98% did not stain for keratin 14-related protein, 54% had significant numbers of AB-PAS-stained cytoplasmic granules, and 16% were identified as ciliated cells. The GS I-negative fraction had a lower colony-forming efficiency than the GS I-positive fraction but, it too, was able to repopulate denuded tracheal grafts with a complete mucociliary epithelium. These results show that both GS I-positive and -negative cells had the potential to proliferate and differentiate into the major tracheal cell types.

Pentamidine: an inhibitor of interleukin-1 that acts via a post-translational event.

Pharmacologic inhibition of cytokines, particularly interleukin-1 (IL-1), potentially has numerous therapeutic applications in inflammatory diseases. We demonstrate that pentamidine, an aromatic diamidine currently used to treat Pneumocystis carinii pneumonia, is a specific and effective inhibitor of cellular IL-1 release from macrophages, and we have shown that this blockage occurs at neither the transcriptional nor the translational level. Pentamidine induced inhibition of IL-1 occurs via an alteration in the post-translational modification of the protein, altering the intracellular and/or membrane cleavage of the 31-kDa pro-IL-1 to the 17-kDa secreted form. In addition, pentamidine exhibited less broad immunosuppressive actions when compared to a corticosteroid, the classical therapeutics utilized for inhibition of cytokine production.

Fetal hematopoietic alterations after maternal exposure to ethylene glycol monomethyl ether: prolymphoid cell targeting.

Ethylene glycol monomethyl ether (EGME), which is extensively used in the chemical industries, has been associated with hematologic disorders in both humans and experimental animals. EGME is metabolized to the active compound methoxy-acetic acid (MAA), which readily crosses the placenta and impairs fetal development. However, little is known about the effect of maternal EGME exposure on the development of fetal immunity. In the present report, in utero treatment with EGME was found to alter expression of murine thymocyte and liver fetal cell-surface markers. Pregnant mice were exposed to 100, 150, or 200 mg/kg EGME from gestational days (gd) 10 to 17 and offspring examined on gd 18. Significant thymic atrophy and cellular depletion were found in EGME-exposed fetal mice. Flow cytometric analysis indicated that EGME treatment resulted in decreased percentages of CD4+8+ thymocytes and increased percentages of CD4-8- thymocytes. In vitro exposure to MAA did not result in decreased thymocyte viability or proliferation. These data suggest that EGME, in addition to producing thymic hypocellularity, may inhibit thymocyte maturation. EGME also reduced the percentage of CD45+ leukocytic cells present in fetal liver, an alteration that appeared to be largely manifested by decreased numbers of CD45R+ and CD44dim prolymphoid cells. In vitro MAA exposure of fetal liver cells enriched for lymphoid precursors resulted in significant inhibition of proliferation. Reconstitution of irradiated hosts with gd 18 fetal liver cells from vehicle and EGME-exposed syngeneic donors demonstrated impaired ability of the EGME-treated fetal liver to repopulate the host spleen with B or T lymphocytes. These data suggest that EGME-induced immunosuppression may result from targeting of multiple hematopoietic compartments. Further, the present data indicate that fetal liver prolymphocytes may represent sensitive targets of EGME exposure.

Topical application of T-2 toxin inhibits the contact hypersensitivity response in BALB/c mice.

T-2 toxin, a trichothecene mycotoxin, has previously been shown to alter immune functions and promote skin tumors. We demonstrate that topically applied T-2 toxin reduces the ear swelling response to oxazolone challenge in BALB/c mice. For this reduction in ear swelling to occur, toxin application must be at, or within, 1 h after challenge. Dose-response studies showed a 44% reduction in ear swelling with 30 ng of T-2 toxin as compared with a similar reduction with 300 ng of dexamethasone. T-2 toxin did not affect Ag transport from the challenge site to the draining lymph nodes as measured by FITC transport. However, T-2 toxin significantly reduced both MHC class II (Ia) expression and Ag presentation at the same concentrations. Because T-2 toxin, a known protein synthesis inhibitor, was found to inhibit protein synthesis in epidermal cell cultures as measured by [3H]-leucine incorporation, cycloheximide was also examined. Cycloheximide reduced both oxazolone-induced ear swelling and Ag presentation in a similar manner to T-2 toxin. One mechanism of action for T-2 toxin in reducing the contact hypersensitivity response is via inhibition of protein synthesis and effective Ag presentation by epidermal Langerhans cells. This may involve alterations in Ia Ag expression, although a role for class II in the induction phase of the contact hypersensitivity response has not been established definitively.

Exposure to tetrachlorodibenzo-p-dioxin (TCDD) alters fetal thymocyte maturation.

We previously reported that thymic atrophy and reduced thymic cellularity associated with prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice are characterized by quantitative alerations in the number of thymocytes expressing CD4 and CD8 surface antigens. In the present study, these observations have been extended to establish the specific thymocyte maturation processes affected by TCDD through an examination of cell size distributions, alpha beta and gamma delta T cell receptor (TCR) expression, peanut agglutinin (PNA) binding, and J11d marker analysis in murine thymocytes exposed prenatally to TCDD. Pregnant mice were administered vehicle, 1.5 or 3.0 micrograms/kg body wt TCDD by gavage on gestational Days (gd) 6-14. Flow cytometry analysis of gd 18 fetal thymocytes revealed a reduction in the number of small CD4+CD8+ double positive (DP) and PNA+, small thymocytes in the TCDD-exposed groups. The large cell population was reduced by TCDD to approximately 70% of control values. There was also a significant shift in TCR expression of thymocytes with a decrease in alpha beta TCR and a concommitant increase in gamma delta TCR expression from TCDD-exposed fetuses. The CD4-CD8+J11d+ thymocytes were increased in TCDD-treated mice while the more mature CD4-CD8+J11d- thymocyte numbers were similar to controls. Taken together, these data indicate that TCDD inhibits thymocyte maturation at the transition phase between the CD4-CD8+J11d+ phenotype and the DP/J11d+ thymocytes.

Development of human lymphocyte-engrafted SCID mice as a model for immunotoxicity assessment.

Mice with the severe combined immunodeficient (SCID) and triple-deficient (bg/nu/xid) mutations lack select populations of functional immune cells. Studies by several laboratories have demonstrated the ability to restore certain missing immune components in these mice by reconstituting with various lymphoid tissues including peripheral blood lymphocytes (PBL) from mice and humans. Such a model could provide an opportunity to examine human lymphoid cells in an in vivo environment for immunotoxicity assessment. In the present studies, bg/nu/xid and SCID mice were reconstituted by intraperitoneal or intravenous injection with either tetanus-immunized syngeneic mouse splenocytes (mo-SPL) or tetanus-immunized human PBLs (hu-PBL) under various test conditions. Hu-PBL-SCID mice from the C.B-17 strain produced more successful human engraftments than mice from the bg/nu/xid or C3H-SCID strains. Using optimal conditions, mo-SPL-SCID and hu-PBL-SCID mice were engrafted and administered either 2,3,7,8-tetrachlorodibenzo-p-dioxin or cyclosporin A (Cys A) and periodically bled to measure tetanus-specific antibody and class-specific immunoglobulin concentrations. Comparison of the chemical-related changes in immunoglobulin and tetanus antibody concentrations revealed some similarities between control mice and mo-SPL-SCID or hu-PBL-SCID mice, particularly with Cys A groups. However, under the various conditions examined, hu-PBL-SCID mice demonstrated considerable variability in their ability to provide consistent reconstitution, thus, limiting the ability to determine whether human cells are more or less susceptible than mouse cells to the test agents. Provided that this system can be refined to provide consistent reconstitution, hu-PBL-SCID mice may be a promising in vivo model for assessment of potential immunotoxic agents.

Pentamidine isethionate reduces Ia expression and antigen presentation by Langerhans cells and inhibits the contact hypersensitivity reaction.

The mechanism of action of pentamidine isethionate, a diamidino compound used in the treatment of Pneumocystis carinii pneumonia, is unknown. We recently reported that this drug may inhibit the release of inflammatory mediators from alveolar macrophages, which may be associated with its antiparasite activity. As a potential anti-inflammatory agent, we report that topically applied pentamidine reduces ear swelling in the contact hypersensitivity reaction to oxazolone in B6C3F1 mice. The application of pentamidine must occur within 1 h, at the challenge site, to be effective. Topical application appears necessary, because i.v. injection had no effect on reduction of ear swelling. In dose-response studies, a 50% reduction in ear swelling was achieved with as little as 20 micrograms of pentamidine. Pentamidine did not affect Ag transport from the challenge site to the draining lymph nodes, as measured by FITC transport. However, there was a 30 to 40% reduction in epidermal cells expressing Ia Ag from pentamidine-treated mouse ears, compared with control. Ia expression is almost exclusively limited to Langerhans cells in the normal epidermis. This reduction in Ia expression was not due to simple depletion of Langerhans cells by pentamidine, because CD45 expression was unaffected. Concurrent with reduced Ia expression, Ag presentation by pentamidine-treated Langerhans cells was also reduced. Taken together, a mechanism of action for pentamidine in inhibition of the contact hypersensitivity reaction appears to be via a reduction in Ag presentation by decreasing Ia+ Langerhans cells.

Fetal thymic atrophy after exposure to T-2 toxin: selectivity for lymphoid progenitor cells.

Treatment of experimental animals with T-2 toxin has been found to markedly decrease thymic cellularity and to suppress cell-mediated immune function. Although T-2 toxin readily crosses the placenta, little is known about its effect on development of immunity following gestational exposure. In the present report, prenatal T-2 toxin resulted in significant fetal thymic atrophy in mice. In vitro exposure to T-2 toxin resulted in decreased thymocyte proliferation, as well as significant but transient increases in thymocyte viability. Cycloheximide increased thymocyte viability parallel to that seen after T-2 toxin, indicating that enhanced viability after T-2 toxin may be the result of inhibited endonuclease synthesis. These findings suggest that direct cytotoxic effects of T-2 toxin make limited contribution to thymic atrophy production. In support of this conclusion, in vivo T-2 toxin exposure resulted in only limited alteration of thymocyte development, as evidenced by expression of CD4, CD8, and alpha beta TCR cell-surface antigens. These data further indicate that antiproliferative effects of T-2 toxin on thymocytes may contribute limitedly to thymic atrophy observed in vivo. In vivo T-2 toxin treatment did not affect total numbers of CD44+, CD45+, or Mac-1+ fetal liver cells. However, such exposure resulted in significant decreases in CD44lo and CD45lo fetal liver prolymphoid cell subpopulations. Subsequent in vitro T-2 toxin exposure of fetal liver cells enriched for lymphoid precursors resulted in both decreased cell viability and highly significant decreased proliferation. Taken together, these data suggest that lymphocyte progenitors, in contrast to thymocytes, represent highly sensitive targets of T-2 toxin exposure, responsible for thymic atrophy.

Thymocyte injury after in vitro chemical exposure: potential mechanisms for thymic atrophy.

In addition to hepatic injury, thymic atrophy is a common observation in rodent subchronic toxicity studies. We have examined representative chemicals which produce thymic atrophy in rodents for their ability to cause direct thymocyte injury because the mechanism(s) responsible for these effects have not been determined. Although a number of the compounds examined failed to have any observable direct effect on thymocytes, others either inhibited lymphocyte proliferation or initiated cell death. In the latter group, thymocyte death was always preceded by increases in intracellular Ca++ and involved, to varying degrees, necrotic and apoptotic events. Apoptosis, as evidenced by cellular DNA cleavage into multiples of 180-200-base pair oligonucleotides and partial cell protection by cycloheximide treatment, was most evident after treatment with acetaldehyde or dibutyltin dichloride. A number of compounds that produce thymic atrophy also inhibited T lymphocyte proliferation without evidence of cell death. Considering that many of the compounds tested failed to produce any evidence of direct thymocyte injury (i.e., necrosis, apoptosis or inhibition of cell proliferation), indirect mechanisms may also be involved in thymic atrophy and may target prothymocytes in the bone marrow, after normal homing patterns or injure the thymic epithelium. Thus, it appears that a variety of mechanisms may be responsible for chemical-induced thymic atrophy and/or injury.

Selective prothymocyte targeting by prenatal diethylstilbesterol exposure.

Estrogens have been reported to modulate immunologic responses at both physiologic and pharmacologic concentrations. Treatment of experimental animals with the synthetic estrogen, diethylstilbesterol (DES), markedly decreases thymic cellularity, manifested histologically as a progressive loss of cortical thymic lymphocytes. In the present report thymic atrophy after prenatal DES exposure was found to be more severe than has been reported following adult exposure, indicating a possible greater sensitivity of the developing immune system to estrogenic hormones. DES exposure resulted in a limited alteration of cell maturation within the fetal thymus as evidenced by only slight alterations in the expression of CD4 and CD8 cell-surface antigens. To examine the possibility that DES targets hematopoietic stem cells in the fetal liver, cytometric analysis was conducted using a panel of fluorescent antibodies to quantitate the hematopoietic subpopulations present in control and DES-exposed Gestational Day (gd) 18 fetal mouse liver. There were no significant DES-induced alterations in the number of hematopoietic stem cells, or in fetal liver cells expressing CD44 (hematopoietic precursors), Mac-1 (granulocyte-macrophage lineage precursors), or CD45R (B-lineage lymphocytes) surface antigens. However, DES selectively reduced the number of fetal liver precursors containing the lymphocyte stem cell-specific DNA polymerase, terminal deoxynucleotidyl transferase, which suggested that DES may specifically target the fetal liver prothymocyte. Reconstitution of irradiated hosts with gd 18 fetal liver from vehicle and DES-exposed syngeneic donors demonstrated an impaired ability of the DES-treated fetal liver to repopulate the thymus of irradiated hosts. In addition, fetal liver cells enriched for prelymphoid cells contained potentially significant levels of estrogen specific receptors. Taken together these data, in conjunction with the lack of direct thymocyte injury (necrosis, apoptosis, and/or inhibition of cell proliferation) by DES treatment, suggest that estrogen-mediated thymic atrophy may result, at least in part, from a specific alteration in the lymphocyte stem cell population responsible for colonizing the thymus.

Organ-specific hematopoietic changes induced by a recombinant human interferon-alpha in mice.

Interferon-alpha (IFN-alpha) is a naturally occurring cytokine that mediates numerous biological activities and has demonstrated therapeutic potential in a variety of malignancies. Encouraging activity against HIV-1 replication has also been observed with IFN-alpha in the treatment of AIDS, although hematotoxicity has been a frequently observed side effect. In addition, in vitro studies have suggested that IFN-alpha may function as a down-regulator of myelopoiesis. A recombinant hybrid of subtypes of human IFN-alpha, rHuIFN-alpha A/D, has antiviral activity in murine cells in vitro and in vivo. This study examines the effect of acute and subchronic exposure to rHuIFN-alpha A/D on hemopoietic and immune parameters in C57Bl/6 mice. IFN-alpha was administered ip at 0, 1000, 10,000, and 100,000 units/day for either 1 or 10 consecutive days. Many of the known effects of IFN-alpha in humans such as anemia, leukopenia, and thrombocytopenia were observed in mice following subchronic exposure, with the latter two effects also manifested following acute exposure. Further analysis showed that this leukopenia was not selective. Both splenic and bone marrow cells were examined following 10 days of dosing with the high dose of IFN-alpha. Lymphocytes were reduced in both compartments, while granulocytes were increased in both compartments. Bone marrow cells programmed to differentiate into granulocytes (CFU-G) were suppressed, while macrophage progenitors (CFU-M) were stimulated. Erythroid cells decreased in the marrow but increased in the spleen, suggesting that the microenvironment may play a significant role in the effect of IFN-alpha. The proliferative capacity of both B and T splenic lymphocytes was significantly suppressed in a dose-related fashion following multiple exposure to IFN-alpha. Clinically, IFN-alpha is most often given in multiple doses and the present data suggest that such a regimen is toxic to both erythroid and myeloid cells, as well as being immunotoxic to splenic B and T lymphocytes.

Perinatal thymocyte antigen expression and postnatal immune development altered by gestational exposure to tetrachlorodibenzo-p-dioxin (TCDD).

In utero exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was found to alter expression of murine thymocyte fetal cell-surface markers. Pregnant mice were treated (via gavage) with 0, 1.5, or 3.0 micrograms TCDD/kg/day in corn oil on gestational days (gd) 6-14. Offspring were examined on gd 18 and postnatally on d6, d14, and d21, and at 7, 8, and 10 weeks of age. Severe thymic atrophy and cellular depletion were found both pre- and postnatally in TCDD-exposed mice. Immunocytochemical localization of the Thy 1.2 antigen on gd 18 thymocytes revealed no TCDD-related changes in cellular distribution. Flow cytometric analysis, however, indicated that the TCDD treatment resulted in a significant decrease in the percentage of CD4+8+ fetal thymocytes, as well as significantly increased CD4-8- and CD4-8+ thymocytes. The increased CD4-8+ population after TCDD was not from induction of Ts cells. At 7-8 weeks postnatally, no differences existed between control and treatment groups in mitogen responses and antibody plaque response. However, altered thymocyte antigen expression was found to correlate with altered postnatal immune function, as evidenced by decreased cytotoxic T lymphocyte response at 8 weeks of age. Taken together, these results indicate that immunosuppression following prenatal exposure to TCDD can be readily detected by qualitative and quantitative changes in the cell surface phenotype of fetal thymocytes. Furthermore, the observed altered distribution suggests that TCDD inhibits normal thymocyte maturational processes.