Macrophage induction of T-suppressor cells in pesticide-exposed and protozoan-infected mice.

The use of infectious pathogens has allowed the detection of the development of synergism between pathogens and ubiquitous environmental chemical contaminants. This synergism has been demonstrated to result in a state of immunosuppression which either did not occur in the independent and singular presence of the chemical or pathogen and/or was greater than additive when both were combined. The immunosuppression was distinct with regard to the organochloride used and, therefore, is not a ubiquitous characteristic of all organohalides. The production of a macrophage soluble factor which appeared to induce T-suppressor cells was demonstrated in hepatic Kupffer cells from mice administered 5 ppm of dieldrin for 10 weeks and then infected with Leishmania tropical promastigotes. The factor was not generated in mice administered dieldrin and infected with malaria nor in mice administered only dieldrin nor in mice only infected with Leishmania. Additional studies revealed a profound impairment in macrophage antigen processing with macrophages obtained from mice administered dieldrin. The use of pathogen models may allow the immunosuppressive potential of environmental chemical contaminants to be expressed in a more sensitive manner.


Introduction
Previous studies have demonstrated that environmental chemical contaminants impair host defense responses to a malaria challenge and to gram-negative endotoxin (1). In conjunction with these alterations, a significant reduction in antibody formation to sheep erythrocytes, (SRBC), a T-dependent and macrophage-processed antigen, was observed (2). These functional alterations were demonstrated in the absence of any lymphoid histopathologic changes. Further studies to examine cell-mediated immunity (CMI) in the xenobiotic treated mice revealed an essentially normal CMI responsiveness as measured by the graft-versushost assay, mixed lymphocyte culture reaction, *Albany Medical College, Albany, New York 12208. Present address: Department of Pharmacology, Pfizer Central Research, Groton, Connecticut 06340. February 1982 polyclonal mitogen activity and lymphocytotoxicity response (3). Since it was felt that the CMI parameters which were measured and which were unaltered were, in general, macrophage-independent whereas the response to malaria, endotoxin and SRBC were macrophage-dependent, the cellular lesion responsible for the impaired malaria, endotoxin and SRBC responses could reside at the level of the macrophage.
Measurements of macrophage affector activity, i.e., cellular respiration, phagocytic activity, phagocytic capacity and chemotaxis revealed no alteration in these parameters (4). However, macrophage effector activity, i.e., antigen processing and tumor cell killing were significantly impaired (5). Therefore, it was speculated that if the cellular lesion responsible for the xenobiotic-induced immunosuppression was at the level of the macrophage, that the lesion could be due to: a functional impairment in the macrophage, induction of a population of suppressor macrophages, production of suppressor 89 factors from the macrophage or a combination of one of the above.
Therefore, further studies were conducted to determine the contribution of the macrophage to xenobiotics-induced immunosuppression. The possible development of a synergistic reaction between the influence of the xenobiotics on the immune response and a concomitant protozoan infection was ascertained concurrently.

Animals
Male BALB/c mice (18-20 g) were used throughout all studies. Chemicals Dieldrin (Shell) and hexachlorobenzene (HCB, Eastman) were administered in the diet at 1 and 5 ppm and 5 and 100 ppm, respectively. Control mice received untreated powdered Wayne Lab Blox. Food and water were provided ad libitum, and a 12:12 day:night photoperiod was maintained. Dietary administration of the test chemicals was for 3, 5 or 10 week periods.

Parasites
Plasmodium berghei (NYU-2) was passaged weekly by intraperitoneal (IP) injection of 1 x 106 infected erythrocytes. The survival time of infected mice injected with this strain of murine malaria at the stated inoculum was approximately 10-12 days. Leishmania tropica was initially derived from a biphasic blood agar medium and then adapted to a defined culture medium consisting of MEM 199 with 10% heat inactivated fetal calf serum. Subcultures were made weekly. The parasite stage used was a promastigote in a midto late-log phase. Survival time of BALB/c mice injected intradermally (ID) with 1 x 106 promastigotes was approximately 15-20 weeks.

Antibody Formation
Antibody production was assessed using the direct Jerne and Nordin (6) plaque assay as previously described (2). Plaque formation to polyvinylpyrrolidine (PVP, Sigma), a T-independent antigen, utilized passively sensitized SRBC coated with PVP by the method of Boyden (7). Preliminary studies revealed that the kinetics of splenic plaque formation in experimental and control mice were compa-90 rable and, therefore, only day 4 was used to subsequently enumerate plaques.

Mononuclear Spleen Cell Populations
Splenic T-and B-lymphocytes were enumerated as previously described (8). T-cells were detected by the presence of theta antigen (Thy-1) and subsequent cytotoxicity induced by anti-theta serum. B-cells were detected by the presence of surface immunoglobulin (Ig) using fluorescein-tagged, goat anti-mouse Ig. Splenic macrophages were stained by the method of Yam, Li and Crosby (9), which detects the presence of surface neutral esterase, a cytochemical marker for macrophages.

Lymphocyte Blastogenesis
Spleens were removed aseptically from mice, controls and experimentals, at the stated intervals (see Tables 5-8), and teased apart in cold RPMI 1640 and strained through a lOxx silk cloth. This single cell suspension was then passaged through nylon wool (Leuko-Pak leukocyte filter, Fenwal Labs, Deerfield, Ill.) which was soaked in saline for 2 hr at 37°C then rinsed three times in doubly distilled water for 3 days, autoclaved and then dried. A 60-ml disposable syringe barrel was then packed with 6 g of nylon wool and then washed with 20 ml of RPMI 1640 containing 5% fetal calf serum. The columns were then drained and placed in a 37°C incubator for 1 hr prior to being loaded with 108 spleen cells in 20 ml of RPMI 1640. The column, with the cells, was incubated at 37°C for 45 min prior to eluting the cells at a 1 ml/min rate. The T-cell enriched suspension was then concentrated by centrifugation at 250 g for 15 min. Cell viability, as determined by trypan blue dye exclusion, was > 85%, and > 95% of the cells were theta positive. The enriched T-cell population was then dispensed in 200 ,ul volumes containing 4 x 105 cells into Linbro flat-bottomed microtest plates (Flow Lab). The RPMI 1640 medium was supplemented with 2mM L-glutamine, 10% fetal calf serum, 50-100 units of penicillin and 50-100 pug of streptomycin/ml. In addition, phytohemagglutinin (PHA-M, B grade, Difco) was added at a concentration of 0.1 ,ug/well. The plates were incubated at 37°C in humidified 5% CO2 for 48 hr, after which 0.1 ,uCi of 3H (specific activity 5 Ci/mmole) was added and the plates incubated for an additional 16 hr prior to harvest. Cell harvesting was conducted as previously described (3), and data are presented as cpm x i03. Antitheta serum treatment of the nylon wool enriched T-cell preparation utilized a 1:80 dilution of anti-Thy-1 antiserum (Cedarlane Labs) and a 1:10 dilution of Environmental Health Perspectives Low-Tox rabbit complement incubated together for 30 min at 37°C. Mitomycin C treatment consisted of incubating the nylon wool passaged cells (107/ml) with 20 ,ug of mitomycin C (Sigma) for 25 min at 37TC. The cells were washed three times in RPMI 1640 prior to use in blastogenesis studies (3).

Macrophage Assay
Alveolar, splenic and peritoneal macrophages were isolated as previously described (4). Briefly, alveolar macrophages were isolated by lavage of the lung with saline, peritoneal macrophages were isolated by saline lavage of the peritoneal cavity, and splenic macrophages were isolated by surface adsorption. Hepatic Kupffer cells were isolated by partial enzymatic digestion of the liver as described by Stege et al. (10). The macrophage preparations were incubated overnight in RPMI 1640 plus 10% FCS in a 5% C02:humidified atmosphere at a cell concentration of 1 x 107 cells/culture plate. The following day the cell supernatant was decanted off and centrifuged at 250 g for 20 min to remove cell debris. A 50 ,ul aliquot of this supernatant was then added to the T-cell blastogenesis cultures, replacing 50 RIl of the RPMI 1640 medium. PHA-induced blastogenesis studies were then conducted as described above.
Macrophage antigen processing of SRBC, a Tdependent antigen which requires macrophage cooperation for immunogenicity, was conducted as previously described (5).

Statistics
All data are reported as the mean with significance denoted by an asterisk where p < 0.05. Significance was determined by using the Student's t test. Significance for mortality data was calculated using the time-effect evaluation of Litchfield (11).

Results
As can be seen in Table 1, the addition of either 1 or 5 ppm of dieldrin to the diet of mice exacerbated the lethality induced by a challenge infection with either the malaria parasite Plasmodium berghei (NYU-2) or Leishmania tropica. The lethality synergized by dieldrin and the parasites appeared to be both time and dose related. However, mice administered 5 or 100 ppm of HCB and then challenged with malaria or Leishmania expressed a synergy only with Leishmania and not malaria. The mortality induced by Leishmania was more marked in dieldrin-exposed mice than in HCB-exposed mice.
When the dieldrin and HCB-treated mice which were infected with malaria or Leishmania and then evaluated for their ability to produce antibody to SRBC, a suppressive synergism was noted primarily in HCB-treated mice ( Table 2). In these mice, the splenic plaque forming response was more suppressed in the combination of HCB + infected mice than in those only HCB-treated or only infected. In dieldrin-treated mice, a synergism was observed only in mice which received 5 ppm of dieldrin plus infection with Leishmania.
Although a suppressed SRBC response was noted in HCB-treated mice infected with either malaria or Leishmania, a decreased antibody formation to PVP, a T-independent antigen, was observed only in Leishmania-infected mice exposed to dieldrin or HCB. Malaria did not act synergistically with dieldrin or HCB to elicit an impaired response to PVP ( Table 3). The decreased antibody formation to PVP in dieldrin-and HCB-treated mice infected with Leishmania was time-and dose-related in dieldrin-treated mice but was only time-related in HCB-treated mice.
An examination of the number of splenic T-cells, B-cells and macrophages revealed no consistent significant alterations in the cell types in either aMale BALB/c mice (18-20 g) were administered dieldrin or HCB at 1 and 5 or 5 and 100 ppm, respectively, for 3, 6 or 10 weeks and then challenged with either 1 x 105 Plasmodium berghei (NYU-2) infected erythrocytes (IP) or 1 x 106 Leishmania tropica promastigotes injected ID.
bMean survival time (days) is denoted for experimental and control mice and significance is indicated by an asterisk where p < 0.05; n = 12 per group. Following injection of the parasite(s) all dietary administration of HCB or dieldrin ceased.  Table  4). The only statistically significant alteration occurred in the 5 ppm dieldrin-treated plus Leishmania-infected mice at the 10 week exposure period in which a significant decrease in macrophages, T-cells and B-cells was observed as compared to normal controls, infected animals, or 5 ppm dieldrin at the 10 week exposure period. In contrast to the apparent absence of any consistent alteration in T-cell numbers in chemical-treated plus infected mice, a significant depression in HCB and dieldrin-treated plus Leishmania-infected mice to the T-cell mitogen, PHA, was demronstrated (Table 5). Isolated splenic T-cells from Leishmaniainfected mice which had been administered dieldrin or HCB had a significantly depressed response to the polyclonal T-cell mitogen PHA. Neither dieldrin or HCB alone or malaria or Leishmania infection alone caused any change in the splenic T-cell enriched population response to PHA. Furthermore, malaria infection in dieldrin or HCB-treated mice post-immunization and to mice infected with L. tropica (1 x 106 promastigotes, ID) on day 20 post infection.
Environmental Health Perspectives aMononuclear splenic cells (T-cells, B-cells, and macrophages) as mean percentages; n = 12. Cellular populations were enumerated on day 4 in malaria-infected mice (P. berghei, 1 x 105, IP) and day 20 in Leishmania-infected (1 x 106 promastigotes, ID) mice. did not alter PHA responsiveness. The dieldrin + Leishmania-induced impairment in PHA activity was time-and dose-related, whereas the change seen in HCB + Leishmania-infected animals was only time-related, i.e., exacerbation with extended dietary administration of HCB.
To further evaluate the impaired PHA responsiveness in dieldrin-treated mice infected with Leishmania, the splenic T-cell enriched population was subjected to mitomycin C and anti-theta (anti-T hy-1) serum. Mitomycin C and anti-theta serum abolished the mitogenic response to PHA, as measured by 3H-thymidine incorporation. When splenic T-cells taken from dieldrin + Leishmania infected mice were added at one-half the normal cell concen-February 1982 tration (4 x 105) to normal (control) splenic T-cells a significant inhibition was demonstrated (Tables 6  and 7. This inhibition could be blocked by pretreating the dieldrin + Leishmania-infected mouse derived T-cells with mitomycin C which suggested an active cell-mediated suppressor. Similarly, when the splenic T-cells from the dieldrin + Leishmania-infected mice were pre-treated with anti-Thy-1 antiserum their inhibitory or suppressive effect was also abolished. These data suggested the presence of a Thy-1 positive suppressor cell in the spleen of the dieldrin-treated/Leishmania-infected mice. Since the Leishmania promastigote has been described as a parasite infecting and proliferating in macrophages it was subsequently decided to de-  aCell preparations were used as previously described in Table  5. The enriched T-cell population at a concentration of 1 x 107 cells/ml was incubated with 20 ,ug/ml of mitomycin C (Sigma) for 25 min at 37°C. The cells were washed three times with fresh medium and numbers adjusted for subsequent use. Each study was conducted in triplicate.
termine if macrophage dysfunction contributed to the induction of the Thy-1 positive suppressor cells. Hepatic Kupffer cells, alveolar, splenic and peritoneal macrophages isolated from dieldrin treated (5 ppm, 10 wk) Leishmania-infected (day 20) mice were cultured for 24 hr, after which their supernatant, following centrifugation at 250g at 4°C for 15 min, was added to cell cultures simultaneously with PHA. Supernatant from hepatic Kupffer cells, and to a lesser extent from splenic macrophages, but not aveolar or peritoneal macrophages, when added 94 aAll cells were prepared as described. Nylon-wool passaged spleen cells were incubated with a 1:80 dilution of anti-thy-l antiserum (Cederlane Labs) and a 1:10 dilution of rabbit complement.
to either control T-cells or T-cells from Leishmaniainfected mice did not alter their response to PHA, however, when added to T-cells from dieldrinor HCB-treated mice or from dieldrin/Leishmaniainfected mice, the supernatant suppressed the T-cell response to PHA (Table 8). Supernatant from macrophage populations from dieldrin-treated mice or Leishmania-infected mice did not alter the response to PHA. The suppressive activity was removed from the supernatant when dialyzed against phosphate-buffered saline overnight at 4°C. The production by hepatic Kupffer cells, and to a lesser extent splenic macrophages, of a suppressive Environmental Health Perspectives factor which impaired T-cell responses to PHA was correlated with the ability of hepatic macrophage from dieldrin/Leishmania treated mice to process an antigen and to transfer an adequate immunogen to a recipient control animal. Similar alterations were observed with splenic macrophages but to a lesser degree (Table 9). The impaired antigen processing capability was correlated with dietary exposure time.

Discussion
Extensive interest in immunotoxicology has been generated within the past 5-6 years by the demonstration that various environmental chemical contaminants may, in laboratory situations, be immunosuppressive. However, apart from contact dermatitis, clinical evidence indicating environmental chemical-induced immune alteration has not been available; although several examples of autoimmune diseases generated by chemical exposure have been reported (12,13).
Experimental immunotoxicologic data have primarily focused on evaluating the immunosuppressive effect of various environmental chemical aMacrophages were isolated from dieldrin (5 ppm)-and HCB (100 ppm)-treated mice infected with malaria or Leishmania on days 4 and 20, respectively. SRBC were opsonized in 50% FCS for 30 min and labeled with Na5lCrO4. A standardized inoculum of 51Cr-SRBC was injected IV into control BALB/c mice and PFC/106 cells were determined on day 4; n = 10. 95 contaminants, food additives, drugs and physical agents while, in general, disregarding the possible immunostimulation that may occur. In this regard, time and dose may be the critical determinants deciding whether a chemical contaminant is considered to be an immunostimulant or immunosuppressant. Furthermore, the catabolic capability of the lymphoid-macrophage elements at both a basal level and at an immune and/or stimulated level have been neglected.
Since the immune response of an organism is a complex multicellular and humoral event an evaluation of a chemical as an immunosuppressant should initially encompass an immune parameter which is a maximal determination of the host immune capacity. Such a measurement is best reflected in the assessment of the host defense using a challenge with a pathogen. The pathogen used may be a bacterium, virus or complex parasite. Indeed, a systematic evaluation of cell-mediated immunity and humoral immunity may be conducted using a series of various pathogens.
In addition to microbial pathogens, suitable parasitic models are also available. Commonly used parasites for immunological studies are: Schistosoma mansoni, Trichinella spiralis, Plasmodium berghei, Toxoplasma gondii, Trypanosoma cruzi, and Leishmania tropica. The present study utilized two of these parasites, Plasmodium berghei (NYU-2) and Leishmania tropica, to evaluate the influence of two common environmental chemical contaminants dieldrin and hexachlorobenzene (HCB) on host defense capacity and immune competence. Plasmodium berghei (NYU-2) is a lethal murine malaria which evokes a T-cell, B-cell and macrophage response. Although it is lethal, survival time is a reflection of immune competence. The L. tropica promastigote is a macrophage parasite which may or may not survive and proliferate in tissue macrophages depending on their functional status (14).
In the present study host defense mechanisms were more significantly impaired in dieldrin-treated mice than in mice receiving HCB. Indeed, the Leishmania model did appear to be a more sensitive indicator of immunotoxicity since mice bearing this parasite had a marked lethality whether treated with dieldrin or HCB (Table 1). However, immunosuppressive synergism to a T-dependent antigen, SRBC, in the parasite and chemical-treated mice was more evident in mice exposed to HCB than to dieldrin ( Table 2). It appeared that the chemical and not the parasite was the determining factor in the antibody response to SRBC. However, when a T-independent antigen was used, i.e., PVP, the only statistically significant immunosuppressive synergism occurred in dieldrin and HCB-treated mice 96 bearing the Leishmania parasite. Since the antibody response to PVP has been reported to be macrophage-independent (15) it would suggest that B-cells/plasma cells are the target cell type involved. Lack of an altered response to T-independent antigens in malaria infected mice has been reported. Since a significant decrease in splenic T-cells, B-cells and macrophages was only seen in the dieldrin/ Leishmania treated mice in the 5 ppm-10 week group, a change in gross cellularity would seem not to be a causal factor. However, a functional alteration can obviously not be excluded.
In this regard, the marked synergism between Leishmania in both dieldrinand HCB-treated mice in regard to their response to PHA denotes a very selective cellular impairment in T-cell functionality. Since the 5 ppm-10 wk dieldrin/Leishmania-treated group demonstrated the most consistent alteration in immune parameters, i.e., depressed SRBC response, depressed PVP responses, and decreased T, B and MO numbers and depressed PHA response, this group of animals was examined in greater detail for cellular hypofunctionality.
Treatment of splenic T-cell enriched populations from the dieldrin/Leishmania treated mice with mitomycin C to inhibit DNA synthesis or with antiserum to the Thy-1 antigen blocked their ability to inhibit the PHA response of normal T-cells. The inhibitory or suppressing activity suggested the presence of Thy-1 positive DNA synthesizing T-cells. Since induction of T-suppressor cells by macrophages has been demonstrated (16) the contribution of macrophages from dieldrin/Leishmania to the induction of T-suppressor cells (T.) was investigated.
Isolated hepatic, and to a lesser extent splenic, macrophages produced a dialyzable factor that when added with PHA to splenic T-cell cultures, suppressed PHA stimulation of the T-cells. This macrophage T-cell suppressor factor did not influence normal control cells nor T-cells from Leishmania infected mice. However, when added to T-cells from dieldrin/Leishmania mice or dieldrin or HCBtreated mice it inhibited their proliferative response. The inhibition seen in HCB derived T-cells suggests that the factor may act on organohalideprimed cells. Although activated macrophages have been demonstrated to secrete inhibitory factors (17) previous studies have shown that macrophages from organohalide xenobiotics are not activated (4). Indeed, as observed in Table 9, the inability of the macrophages to present an adequate immunogen to naive hosts indicates a suppressed state.
The demonstration that maximal suppressor factor was found associated with hepatic Kupffer cells may be, in part, related to hepatotoxicity of the two xenobiotics. If this is the case, it may provide a Environmental Health Perspectives valuable tool in the study of the relationship between hepatotoxicity and immunosuppression. Further studies to elucidate the factor produced by the suppressor macrophages will evaluate interferon production as well as prostaglandins which have immunomodulatory roles (18). In addition, macrophage secreted factors may also compete with thymidine for its uptake into DNA by T-cells.
The incorporation of infectious disease models into immunotoxicologic studies may be a more relevant as well as more sensitive indicator of immunotoxicity. Although most studies have utilized mature animals, the use of neonates and aged animals in conjunction with stress may be a more accurate measurement of immune potential.