Antibody Plaque-forming Cell Response Research Articles

N,N,-Diethyl-m-Toluamide (DEET) Suppresses Humoral Immunological Function in B6C3F1 Mice

N,N-diethyl-meta-toluamide (DEET) is a particularly effective broad-spectrum insect repellent used commonly in recreational, occupational and military environments. Due to its widespread use and suggested link to Gulf War Illness, this study examined the immunotoxicity of DEET. Adult female B6C3F1 mice were injected sc for 14 days with DEET at 0, 7.7, 15.5, 31, or 62 mg/kg/day. Due to differences in the dermal absorption of DEET between mice and humans, this study eliminated this confounding factor by utilizing sc injection and measured circulating blood levels of DEET to assess bioavailability from sc administration. Effects on lymphocyte proliferation, natural killer cell activity, thymus and spleen weight and cellularity, the antibody plaque-forming cell (PFC) response, and thymic and splenic CD4/CD8 lymphocyte subpopulations were assessed 24 h after the last dose. No effect was observed in lymphocyte proliferation, natural killer cell activity, thymic weight, splenic weight, thymic cellularity, or splenic cellularity. Significant decreases were observed in the percentage of splenic CD4-/CD8- and CD4+/CD8- lymphocytes but only at the 62 mg DEET/kg/day treatment level and not in absolute numbers of these cells types. Additionally, significant decreases in the antibody PFC response were observed following treatment with 15.5, 31, or 62 mg DEET/kg/day. Pharmacokinetic (PK) data from the current study indicate 95% bioavailability of the administered dose. Therefore, it is likely that DEET exposure ranges applied in this study are comparable to currently reported occupational usage. Together, the evidence for immunosuppression and available PK data suggest a potential human health risk associated with DEET in the occupational or military environments assuming similar sensitivity between human and rodent responses.

Acetone in Drinking Water Does Not Modulate Humoral Immunity in Mice as Measured by the Antibody, Plaque-Forming Cell Assay

It has been reported that the repeated topical, nonoccluded application of acetone may modulate antibody production in mice, thus producing humoral immunosuppression. However, the evaporative loss expected following nonoccluded dermal application of acetone makes the systemic effect seem unlikely. This study was designed to investigate the immunotoxicity potential of acetone in mice following a more direct systemic route of dosing via drinking water for 28 days. CD-1 male mice consumed average daily acetone doses of 121, 621 or 1144 mg/kg/day. The antibody, plaque-forming cell (AFC) assay was performed to measure the T cell-dependent, anti-sheep red blood cell immunoglobulin M (IgM) response, and hematology and thymus weights were evaluated to provide additional insight into the potential effects to the immune system. Body weights, white blood cell (WBC), numbers, red blood cell (RBC) counts, and hemoglobin and hematocrit levels showed no treatment-related effects at any dose of acetone. Eosinophil percentages were variable but also showed no dose-related trends. Spleen and thymus weights were not statistically different from controls and there were no effects on spleen cellularity or AFC response as a result of acetone administration. The AFC responses ranged from 1088 to 1401 AFCs/10(6) splenocytes and were not statistically different from controls (1277 AFCs/10(6) cells). Mice treated with cyclophosphamide (20 mg/kg) on days 25 to 28 demonstrated a 94% reduction in AFC/10(6) cells. Thus, the direct systemic administration of acetone did not produce evidence for immunotoxicity in CD-1 mice and the no observed adverse effect level (NOAEL) in this study was determined to be 1144 mg/kg/day.

Effect of nicotine on the immune system: possible regulation of immune responses by central and peripheral mechanisms.

Nicotine (NT) treatment impairs T-cell receptor (TCR)-mediated signaling, leading to the arrest of T cells in the G1 phase of the cell cycle and inhibition of the antibody plaque-forming cell (AFC) response to sheep red blood cells (SRBC). This paper summarizes some of the previous findings related to cigarette smoke/NT and the immune response, and presents preliminary evidence suggesting that mice chronically treated with NT (0.5 mg/day/kg body weight) have a depressed inflammatory response in the turpentine-induced abscess model of inflammation. This ability of nicotine to attenuate an inflammatory response may also be the cause of reduced mortality of chronically nicotine-treated mice from acute influenza A pneumonitis. Moreover, in LEW rats, decreased anti-SRBC AFC responses were also observed after intracerebroventricular (i.c.v.) administration of relatively small concentrations of NT (28 micrograms/day/kg body weight) which, when given peripherally, did not affect the AFC response. In vitro the addition of NT to T cells increased protein tyrosine kinase (PTK) activity and intracellular Ca2+ concentration [Ca2+]i. These results support the hypothesis that NT alters immune responses by directly interacting with T cells, as well as indirectly through brain-immune interactions.

The effects of perinatal/juvenile methoxychlor exposure on adult rat nervous, immune, and reproductive system function.

In order to address data gaps identified by the NAS report Pesticides in the Diets of Infants and Children, a study was performed using methoxychlor (MXC). Female rats were gavaged with MXC at 0, 5, 50, or 150 mg/kg/day for the week before and the week after birth, whereupon the pups were directly dosed with MXC from postnatal day (pnd) 7. Some dams were killed pnd7 and milk and plasma were assayed for MXC and metabolites. For one cohort of juveniles, treatment stopped at pnd21; a modified functional observational battery was used to assess neurobehavioral changes. Other cohorts of juveniles were dosed until pnd42 and evaluated for changes to the immune system and for reproductive toxicity. Dose-dependent amounts of MXC and metabolites were present in milk and plasma of dams and pups. The high dose of MXC reduced litter size by approximately 17%. Ano-genital distance was unchanged, although vaginal opening was accelerated in all treated groups, and male prepuce separation was delayed at the middle and high doses by 8 and 34 days, respectively. In the neurobehavioral evaluation, high-dose males were more excitable, but other changes were inconsistent and insubstantial. A decrease in the antibody plaque-forming cell response was seen in males only. Adult estrous cyclicity was disrupted at 50 and 150 MXC, doses which also showed reduced rates of pregnancy and delivery. Uterine weights (corrected for pregnancy) were reduced in all treated pregnant females. High-dose males impregnated fewer untreated females; epididymal sperm count and testis weight were reduced at the high, or top two, doses, respectively. All groups of treated females showed uterine dysplasias and less mammary alveolar development; estrous levels of follicle stimulating hormone were lower in all treated groups, and estrus progesterone levels were lower at 50 and 150 MXC, attributed to fewer corpora lutea secondary to ovulation defects. These data collectively show that the primary adult effects of early exposure to MXC are reproductive, show that 5 mg/kg/day is not a NO(A)EL in rats with this exposure paradigm (based on changes in day of vaginal opening, pubertal ovary weights, adult uterine and seminal vesicle weights, and female hormone data) and imply that the sites of action are both central and peripheral.

Evaluation of the effect of low-level 2,3,7,8-tetrachlorodibenzo- p-dioxin exposure on cell mediated immunity

The immunotoxicity of TCDD in the mouse has been well documented. To date, the most sensitive endpoint to TCDD-induced toxicity in mice is that reported by Clark et al. (Clark, D.A., Gauldie, J., Szewczuk M.R. and Sweeney, G. (1981) Proc. Soc. Exper. Biol. Med. 168, 290.) who found that TCDD suppressed the murine cytotoxic T lymphocyte (CTL) response following four weekly doses of 4 ng TCDD/kg/week. However, these results have never been corroborated, as other laboratories have been unable to detect immunosuppression by TCDD at such low levels. In this study, we evaluated the effect of TCDD on the in vivo- and in vitro- generated CTL response to P815 mastocytoma cells in adult C57BL/6J female mice via a 51Cr release assay. Mice were given weekly intraperitoneal injections of TCDD or vehicle for 4 weeks at dosages ranging from 0.01 to 3.00 μg/kg/week. No statistically significant suppression of the in vivo- or in vitro-generated CTL response was detected at any dosage. As expected, significant increases in liver weights and decreases in thymus weights were observed at TCDD dosages of 1.0 and 3.0 μg/kg/week. Likewise, suppression of the antibody plaque forming cell response to sheep erythrocytes was observed at dosages of 1.0 and 3.0 μg TCDD/kg/week. Although expected humoral immunosuppression and organ effects were observed, our data do not support suppression of murine CTL responses at the TCDD doses employed in this study.

Subacute Immunotoxicity Study of Formaldehyde in Male Rats

Immune functions were examined in male rats following 28 day oral administration of formaldehyde by gastric tube at dose levels of 0, 20, 40, and 80 mg/kg. Routine parameters examined included hematology, clinical chemistry, and body, thymus, kidney, and liver weights. In addition, cellularity of spleen and lymph nodes, histology of spleen, thymus, lymph nodes, liver kidney, small and large intestines, and histochemistry of spleen and lymph nodes were evaluated. Immune parameters evaluated included serum hemagglutinin antibody response; antibody plaque forming cell response to sheep erythrocytes (lymphocyte-dependent antigen); microbicidal activity of Candida albicans; and phagocytic activity by adhesion of microspheric hydrophilic synthetic particles to leukocyte cell membrane. Body weights were slightly decreased at high dose level (80 mg/kg). The difference was significant when compared to the controls. The lymph node weights were significantly increased in rats receiving formaldehyde. The cellularity of lymphoid organs was not influenced after 28 day exposure to formaldehyde.

Evidence of altered T-lymphocyte number and proliferative responses in genetically epilepsy-prone rats

Genetically epilepsy-prone (GEPR-9) rats exhibit decreased antibody plaque-forming cell responses following immunization. We examined the hypothesis that this immunosuppression was due to deficits in the number or proliferative responses of T-lymphocytes. Splenocyte responses to concanavalin A and pokeweed mitogen were significantly greater in GEPR-9 rats than controls. Flow cytometric analysis indicated that GEPR-9 rats possess an increase in T-cells associated with the T-helper phenotype. The increased proportion of T-helper cells in GEPR-9 rats may underlie their enhanced proliferative responses to T-cell mitogens. These results clearly indicate that the failure of the GEPR-9 rat to respond to a T-dependent antigen in vivo is not due to a lack of T-helper activity.

Risk assessment in immunotoxicology: I. Sensitivity and predictability of immune tests

We have previously reported on the design and content of a screening battery involving a “tier” approach for detecting potential immunotoxic compounds in mice ( Luster et al., 1988 , Fundam. Appl. Toxicol. 10, 2–19). This battery has now been utilized to examine a variety of compounds by the NIEHS Immunotoxicology Laboratory, the National Toxicology Program-sponsored laboratories, and by the Cell Biology Department at the Chemical Industry Institute of Toxicology. The database generated from these studies, which consists of over 50 selected compounds, has been collected and analyzed in an attempt to improve future testing strategies and provide information to aid in quantitative risk assessment for immunotoxicity. Studies presented here have established the ability of each of the tests or test combinations in the screening battery to detect immunotoxic compounds. Efforts are currently underway using this database to determine the relationships between these immune tests and susceptibility to challenge with infectious agents or transplantable tumor cells. The present analyses indicated that the performance of only two or three immune tests are sufficient to predict immunotoxic compounds in rodents (> 90% concordance). The tests that showed the highest association with immunotoxicity were the splenic antibody plaque forming cell response (78%) and cell surface marker analysis (83%). The relationship between immunotoxicity and carcinogenicity, as well as genotoxicity, was also determined. These analyses suggested that potential immunotoxic compounds are likely to be rodent carcinogens ( p = 0.019) although for compounds that are not immunotoxic the carcinogenic status is unclear. There was no relationship observed between immunotoxicity and mutagenicity as determined using in vitro genotoxicity tests. The significance of these observations is discussed in terms of the relationship between immunotoxicity tests and biological/toxicological processes concerned with human health (e.g., infectious disease).

Risk Assessment in Immunotoxicology

We have previously reported on the design and content of a screening battery involving a “tier” approach for detecting potential immunotoxic compounds in mice (Luster et al., 1988, Fundam. Appl. Toxicol. 10, 2–19). This battery has now been utilized to examine a variety of compounds by the NIEHS Immunotoxicology Laboratory, the National Toxicology Programsponsored laboratories, and by the Cell Biology Department at the Chemical Industry Institute of Toxicology. The database generated from these studies, which consists of over 50 selected compounds, has been collected and analyzed in an attempt to improve future testing strategies and provide information to aid in quantitative risk assessment for immunotoxicity. Studies presented here have established the ability of each of the tests or test combinations in the screening battery to detect immunotoxic compounds. Efforts are currently underway using this database to determine the relationships between these immune tests and susceptibility to challenge with infectious agents or transplantable tumor cells. The present analyses indicated that the performance of only two or three immune tests are sufficient to predict immunotoxic compounds in rodents (>90% concordance). The tests that showed the highest association with immunotoxicity were the splenic antibody plaque forming cell response (78%) and cell surface marker analysis (83%). The relationship between immunotoxicity and carcinogenicity, as well as genotoxicity, was also determined. These analyses suggested that potential immunotoxic compounds are likely to be rodent carcinogens (p = 0.019) although for compounds that are not immunotoxic the carcinogenic status is unclear. There was no relationship observed between immunotoxicity and mutagenicity as determined using in vitro genotoxicity tests. The significance of these observations is discussed in terms of the relationship between immunotoxicity tests and biological/toxicological processes concerned with human health (e.g., infectious disease).

A comparison of three nucleoside analogs with anti-retroviral activity on immune and hematopoietic functions in mice: In vitro toxicity to precursor cells and microstromal environment

A number of 2′3′-dideoxynucleosides have been reported to markedly inhibit the in vitro growth of HIV, the causative agent of acquired immunodeficiency syndrome (AIDS). Clinical trials have shown that the continued therapeutic use of these nucleoside derivatives can be associated with adverse side effects. Since these side effects include myelotoxicity, as occurs in many patients treated with zidovudine (AZT; 3′-azido′3-deoxythymidine), and AIDS patients already represent an immunologically compromised population, we examined the immunological effects of three nucleoside inhibitors, including zidovudine, 2′3′-dideoxycytidine, and 2′3′-dideoxyadenosine (DDA) in a mouse model. Additional studies were conducted to further determine and characterize the potential toxic effects associated with these drugs on the hematopoietic system. Of the three dideoxynucleosides examined, only DDA altered immune functions following a 30-day subchronic exposure in mice. This was evidenced by a marked suppression of the antibody plaque-forming cell response and a slight alteration in macrophage function. None of the nucleoside derivatives affected bone marrow function following in vivo exposure, although AZT produced a mild macrocytic anemia in vivo and was myelotoxic when added in vitro to bone marrow cell cultures. In vitro studies indicated that AZT was capable of affecting both proliferating stem cells as well as the stromal cell microenvironment, both of which play a role in hematopoiesis. These data indicate that, although the mice may not develop the identical toxicities associated with nucleoside therapy in humans, certain adverse immunological and hematological effects were readily discerned which could have relevance to humans.

Selective immunosuppression following exposure to a novel anthraquinone, 1,4-bis[(2-aminoethyl)amino]-5,8-dihydroxy-9, 10-anthracenedione dihydrochloride (AEAD)

The immune response of murine splenic lymphocytes was characterized following both in vivo and in vitro exposure to the novel and highly-substituted anthraquinone AEAD. Gross indicators of toxicity such as changes in body weight, lymphoid organ weights or lymphoid organ cellularity were unaffected. Similarly, the antibody plaque-forming cell response and natural killer cell function were unaltered. The most consistent effect noted was the selective suppression of the cytotoxic T-lymphocyte (CTL) response, which persisted for an extended period of time following in vivo exposure at concentrations of 0.75–3.0 μg/g. In contrast to the CTL suppression induced by the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]-anthracene, AEAD-induced CTL suppression could not be restored by addition of exogenous interleukin-2. Studies are currently underway to further characterize AEAD-induced immunosuppression at the cellular and molecular levels.

Immunological studies on paroxetine, a novel anti-depressant drug

Paroxetine is a novel and selective neuronal 5-hydroxy-tryptamine uptake inhibitor with antidepressant activity. Paroxetine was examined for its ability to induce adverse immunological reactions, either as a consequence of a specific immune response or by a direct or indirect effect on the immune system. Paroxetine did not react in vitro with protein amino or thiol groups, suggesting that it lacks the capacity to form potentially immunogenic hapten protein conjugates. No anti-paroxetine antibody was detected in plasma or serum samples from patients and rats following oral administration over prolonged periods, or from epicutaneously exposed guinea pigs, or from rabbits given paroxetine in Freund's adjuvant, suggesting that paroxetine does not have the capacity to elicit humoral immune responses. Guinea pigs epicutaneously exposed to paroxetine did not develop contact sensitivity, suggesting that it does not have the capacity to elicit cell-mediated immune responses. These results suggest that paroxetine lacks intrinsic immunogenicity. Anti-SRBC antibody plaque-forming cell responses in mice were unaffected by oral administration of paroxetine, and paroxetine had no significant effect on ex vivo and in vitro murine macrophage phagocytosis of opsonized SRBC or on ex vivo murine splenocyte mitogen responses, suggesting that paroxetine does not exert modulatory effects on the immune system or on macrophage function. These findings, together with the results of pre-clinicsl safety evaluation studies, suggest that paroxetine is unlikely to have immunotoxic effects.

Immunomodulatory activity of Panax ginseng extract.

Panax ginseng extract is capable of enhancing antibody plaque forming cell response and circulating antibody titre against sheep erythrocytes, cell mediated immunity against Semliki forest virus antigen, and natural killer cell activity in mice. It also enhanced production of interferon of both the pH stable and labile types induced by an interferon inducer of fungal origin.

Studies of immune function and host resistance in B6C3F1 mice exposed to formaldehyde

A series of immune function and host resistance parameters were examined in female B6C3F1 mice following a 21-day (6 hr/day) inhalation exposure to 15 ppm of formaldehyde (HCHO). Immune parameters examined included delayed hypersensitivity to keyhole limpet hemocyanin, antibody plaque-forming cell response to sheep erythrocytes (T-lymphocyte-dependent antigen) and TNP-Ficoll (T-lymphocyte-independent antigen), lymphoid organ weights and histopathology, routine hematology, bone marrow cellularity and CFU progenitor cell enumeration, lymphocyte subpopulation quantitation by cell surface markers, mitogen-induced lymphocyte blastogenesis, macrophage function parameters, and host resistance to challenge with the bacterium Listeria monocytogenes and transplantable tumor cells. Lymphoid organ weight, bone marrow cellularity, and hematology parameters were unchanged in HCHO exposed mice. Similarly, the percentage of T and B lymphocytes and their proliferative responses to mitogens were not significantly altered. Antibody (IgM) plaque-forming cell response following antigen challenge was unchanged. Macrophage function was normal although some evidence of enhanced H 2O 2 production associated with elevated bactericidal activity was observed in resident macrophages. Resistance to challenge with the bacteria Listeria monocytogenes was significantly enhanced, while resistance to tumor challenge remained unchanged. No evidence of immunosuppression following short-term exposure to HCHO was observed.

Inhibition of the polyclonal antibody plaque-forming cell response of human B lymphocytes by C-reactive protein (CRP) and CRP complexes

The pokeweed mitogen (PWM)-induced generation of polyclonal immunoglobulin-secreting cells (ISC), as measured by reverse hemolytic plaque formation of protein A-coated sheep E by human blood mononuclear cells, was inhibited by both purified human C-reactive protein (CRP) and CRP-C-polysaccharide (CRP-CPS) complexes. CRP and CRP-CPS mediated the suppression by binding and activating monocytes and T cells with IgG Fc receptors. The extent of suppression was dependent on CRP concentration and the CRP/CPS ratio and was similar to that obtained with IgG immune complexes. In contrast, CRP did not alter the number of ISC formed in response to the relatively T-independent polyclonal activator, protein A-bearing staphylococci. Suppression of ISC formation was most likely confined to events associated with the terminal stages of B-cell differentiation since no effect of CRP or CRP-CPS on the blastogenic response to polyclonal B-cell activators (PBA) was detected. These findings indicate that the acute phase reactant CRP has the potential to modulate antibody responses during the course of an inflammatory response.

Suppression of the leukocyte adherence inhibition response to murine-sarcoma-virus-induced tumors by tumor-associated suppressor cells.

The tumor-specific leukocyte adherence inhibition (LAI) response of lymphoid cells from C57B1/6 mice sensitized to a murine-sarcoma-virus (MuSV)-induced tumor was inhibited by adherent phagocytic spleen cells from mice with progressively growing RBL-5 tumors. Both the direct LAI response and the formation of the mediator of LAI were sensitive to the suppressive activity of the progressor spleen macrophages. Suppression of LAI was not restricted by H-2 differences between the sensitized lymphocytes and progressor macrophages. Suppression was also nonspecific since the antibody plaque-forming cell response of normal lymphocytes to triphenylated hemocyanin was lowered by the addition of progressor spleen macrophages. Removal of adherent suppressor macrophages during the late stages of tumor growth revealed greatly elevated LAI reactivity not detectable in an unfractionated population.

Relationship between antigenic stimulation and increased splenic peroxidase levels during the immune response

Immunization of mice with either soluble or particulate antigens induced a marked increase in peroxidase activity of spleen cell homogenates. Animals immunized with sheep erythrocytes showed maximum splenic peroxidase activity at 2 days. By the 4th day after immunization, when the hemolytic antibody plaque-forming cell response was maximal, peroxidase activity returned to normal levels. Increased splenic peroxidase activity also occurred in mice immunized with rabbit erythrocytes, as well as with rabbit serum or bovine serum albumin. No change in splenic peroxidase activity occurred in mice injected with syngeneic mouse erythrocytes or serum. Both glass-adherent spleen cell populations, morphologically consisting of 90 to 95% macrophages, as well as nonadherent cells, consisting of more than 90% lymphocytes by the same criteria, showed peroxidase activity. Immunization of mice with sheep erythrocytes resulted in an intracellular redistribution of the peroxidase activity among several distinct subcellular fractions prepared by differential centrifugation. Maximum redistribution occurred with granule-associated enzyme activity. A possible relationship between peroxidase activity with functional activity of lymphocytes and macrophages during humor immune responses seemed likely.

Strain differences in the antibody plaque-forming cell responses of inbred mice to pneumococcal polysaccharide

Several inbred strains of mice differed in their ability to respond to Type III pneumococcal polysaccharide (SIII) at both the circulating antibody and cellular levels. The PFC response to SIII was qualitatively similar for all strains of mice tested in that only direct PFC were detected. The kinetics of the response were also similar for several of the inbred strains of mice but differed for random-bred mice. The ability of certain strains of mice to respond well to SIII was dominant over low responsiveness and was not associated with the H-2 locus in contrast to the control of responsiveness exerted by this locus for several polypeptide and protein antigens.