Immunological studies on mice exposed subacutely to methyl isocyanate.

The immunotoxicity of methyl isocyanate (MIC) was evaluated in female B6C3F1 mice exposed via inhalation to 0, 1, or 3 ppm for 6 hr per day on 4 consecutive days. The antibody response to sheep erythrocytes and natural killer cell activity were found to be unaffected by MIC exposure. Although lymphoproliferative responses to mitogens were moderately suppressed by MIC, the differences were not statistically significant. The response of splenic lymphocytes to allogeneic leukocytes in a mixed leukocyte response (MLR) was suppressed in a dose-related fashion and was significantly different from the control response at the 3 ppm level. This effect was thought to be secondary and a result of general toxicity, rather than a direct effect of MIC on the immune system. Furthermore, resistance to the infectious agents Listeria monocytogenes, mouse malaria parasite, and influenza virus, or to transplantable tumor cells was not compromised by MIC exposure. Thus, the immune system does not appear to be a primary target for MIC toxicity.


Introduction
Although the lung is recognized as the primary target organ of methyl isocyanate (MIC), the toxicological evaluation recently undertaken by the National Toxicology Program included genetic toxicity, reproductive toxicity, and immunotoxicity studies. Toxicity in these organ systems could result from secondary, if not primary, effects of MIC. In order to maximize any potential of this highly reactive chemical to reach remote tissues, the specialized studies were conducted on animals exposed subacutely. The immunotoxicity studies reported here were composed of selected assays for immune function and host resistance following exposure of female B6C3F1 mice to 0, 1, or 3 ppm MIC for 6 hr periods on 4 consecutive days. With this exposure regimen, 3 ppm represents approximately one-half the LC50. No deaths occurred at 3 ppm, although pulmonary pathology was present. General toxicity was indicated by the lethargic appearance of the mice following exposure, a significantly decreased body weight on the day following the last exposure, and significant decreases in spleen and thymus weights 7 days after the last exposure (1). All but certain lung changes were reversible within 28 days. *National Toxicology Program, National Institute of Environmental Health Sciences, P. 0. Box 12233, Research Triangle Park, NC 27709.

Exposure of Animals
The procedure for exposure of animals to methyl isocyanate (MIC) by inhalation has been described fully (3). Exposures were conducted on 3-11 to 3-14 and 6-17 to 6-20-85. Repeated dose studies were conducted in stainless-steel, wire-mesh exposure cages by exposing 10-week old B6C3F1 female mice 6 hr/day for 4 consecutive days to 0, 1, or 3 ppm MIC. During nonexposure periods, mice were housed in an adjacent room on a 12 hr light cycle with food and water ad libitum. Immunotoxicity tests were initiated within 5 days of the last exposure.

Immune Function Assays
Humoral immunity was measured by the response to sheep erythrocytes, a T-dependent antigen, in mice immunized 1 day after the last exposure. IgM plaque forming cells (PFC) were enumerated in spleen cells 4 days later using a modified Jerne plaque assay in agar (4). Natural killer (NK) cell activity of spleen cells was determined using YAC-1 tumor target cells in a 51Cr-release assay (5). Several effector:target (E:T) ratios were used, but data are reported only from the 100:1 ratio, which gave maximal lysis. Lymphocyte proliferation was measured by quantitating 3H-thymidine incorporation in splenic lymphocytes responding to the T-cell mitogens phytohemagglutinin (PHA) or concanavalin A (Con A), to the B-cell mitogen E. coli lipopolysaccharide (LPS), and to allogeneic leukocytes (mitomycin Ctreated lymphocytes from DBA/2N mice) in a mixed leukocyte response (MLR). Mice were sacrificed 4 days after the last exposure for this assay. Conditions for this assay have been previously published (6). Although several concentrations of mitogens were run, only values from optimal mitogen concentrations are reported.

Host Resistance Assays
Mice were challenged with infectious agents 5 days after the last exposure to MIC. Resistance to bacterial challenge with Listeria monocytogenes (strain L242/73 type 4B) was tested by IV injection of 2 x 104 viable  'NK cytotoxicity against 51Cr-labeled YAC-1 target cells was measured at a 100:1 E:T ratio in two separate experiments. Cytotoxic activity was calculated from the formula: % cytotoxicity -cpmT -SR x 100 in which cpmE -SR is cpm of 5"Cr release in the experimental minus spontaneous release, and cpmT -SR is cpm in total cells added (total release) minus spontaneous release.
b Seven mice per group.
c No values differ significantly from control. bacteria, which approximates an LD20 in control mice. Mortality was monitored for 14 days. A nonlethal strain of malaria, Plasmodium yoelii 17XNL, was administered by IV injection of 1 x 106 parasitized erythrocytes. The percentage of parasitized erythrocytes, determined by Diff-Quik staining of tail blood, was followed through the course of infection. The influenza challenge model used was developed by IIT Research Institute, Chicago, IL, under the direction of James Fenters and Peter Barbera (Report L06133-5 prepared for NIEHS contract N01-ES-1-5000). Influenza A2/Taiwan/64 virus was instilled intranasally, and mortality was monitored for 14 days. Resistance to syngeneic tumor cell challenge was determined by injecting 1 x 104 B16F1O tumor cells IV and determining tumor burden in the lungs 21 days later by quantitation of 125I-iododeoxyuridine (7). Host resistance data from two separate studies was pooled for analysis.

Statistical Analysis
Data from immune function assays were analyzed by the RS/1 multicompare procedure using the Wilk-Shapiro test for normality, one-way analysis of variance, and Dunnett's test for multiple comparison with a control. Mortality data were analyzed by the chi-square test.

Results and Discussion
Toxicology Based on previously reported data, mice subacutely exposed to 3 ppm of MIC exhibited signs of toxicity which included lethargy and decreases in body and certain organ weights, notably spleen and thymus (1). Hematological values, determined 5 days after the last exposure, were normal with the exception of hematocrit values, which were slightly higher in both of the MICexposed groups, perhaps reflecting some dehydration. The transient nature of the observed effects suggested a general rather than a specific systemic toxicological response. However, pathologic changes in the respiratory system were seen, and included erosion and necrosis of the epithelium of the nasal cavity, trachea, and bronchi, fibrosis in the walls of major bronchi, and fibroepithelial tissue projections into major airways (2).

Immune Function
The results of two separate experiments evaluating the antibody response of MIC-exposed mice are shown in Table 1. No effect of MIC was detected on the PFC response to sheep erythrocytes. NK cell activity from two separate MIC exposures was also found to be unaltered by chemical exposure (Table 2). Splenic cellularity was also not altered by MIC exposure (data not shown).
The proliferative response of splenic lymphocytes from MIC-treated mice to B-and T-cell mitogens and allogeneic leukocytes is shown in Table 3. There was a trend toward suppression of the response to mitogens in treated mice and a statistically significant depression of the MLR at 3 ppm. A repeat study gave similar results, with the MLR again suppressed in a dose-related fashion (data not shown). The suppression was transient, however, as mice evaluated at the end of the 120-day observation period were found to be unaffected by chemical exposure (data not shown).

Host Resistance
Following chemical exposure, groups of mice were challenged with either Listeria monocytogenes, mouse malaria (Plasmodium yoelli), influenza virus, or B16F10 syngeneic tumor cells for evaluation of host resistance. The results, representing pooled data from the two separate exposures, are summarized in Figure  1. The Listeria and B16F10 studies included a positive control using cyclophosphamide at 20 mg/kg, which caused significantly impaired resistance in both cases (data not shown). There were no statistically significant alterations in any of the host resistance models as a result of MIC exposure, although there was a trend toward susceptibility following intranasal challenge with influenza virus. This effect may be immunological in nature or a consequence of impairment of lung clearance mechanisms.
In conclusion, the immune system does not appear to be sensitive to MIC exposure in mice. The minimal changes in immune function that were observed are possibly a secondary consequence of the pulmonary or general toxicity observed in exposed mice. In light of the normal response of MIC-treated mice to challenge with infectious agents and tumor cells, the immune alterations must be considered biologically inconsequential. Although MIC did not appear to reach primary organs of the immune system, it could cause alterations in immune function as a secondary consequence to lung disease as has been seen in asbestosis in man (8) and in mice (9). This aspect of MIC toxicity was not evaluated in the present study.