Oral Treatment with Trimethoprim-Sulfamethoxazole and Zidovudine Suppresses Murine Accessory Cell-Dependent Immune Responses

Yvonne R. Freund1, Linda Dousman, James T. MacGregor2 and Nahid Mohagheghpour

SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025-3493

Received November 2, 1999; accepted January 4, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trimethoprim-sulfamethoxazole (TMP-SMX), commonly used for prophylaxis of Pneumocystis carinii pneumonia (PCP) in AIDS patients, often produces a high incidence of treatment-limiting reactions. We investigated the effect of oral administration of TMP-SMX alone or in combination with the antiretroviral drug zidovudine (ZDV) on hematopoiesis and cellular immunity in BALB/c mice. Daily treatment for 28 days with TMP-SMX (160:800 mg/kg) had no effect on hematopoiesis or the ex vivo proliferative response of splenic T lymphocytes to allogeneic tumor cells (EL-4) or to concanavalin A (ConA), or that of splenic B cells to lipopolysaccharide (LPS). ZDV at 240 mg/kg/day was not immunosuppressive but caused a mild macrocytic anemia. Combined treatment produced severe pancytopenia, a significant drop in splenic cellularity, and a 61% decrease in the percentage of splenic macrophages. The percentage of splenic CD3+ lymphocytes increased 150% in the TMP-SMX + ZDV group, but the ratios of T-cell subsets and the frequency of B cells remained unchanged. Combined drug treatment did not impair the proliferative response of B cells to LPS or that of T cells to EL-4 cells. In concert with the reduction in the percentage of macrophages, the proliferative response of T lymphocytes to ConA decreased significantly. Optimal ConA-induced T-cell proliferation requires the participation of accessory cells (AC) (e.g., macrophages); EL-4 cells are able to function as AC. These data indicate that ZDV synergizes with TMP-SMX, causing severe hematotoxicity and suppressing AC-dependent immune function, and suggest that this therapeutic regimen may contribute to the immune deterioration in AIDS patients.

Key Words: trimethoprim-sulfamethoxazole; zidovudine; Pneumocystis carinii pneumonia; immunosuppression; accessory cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Before highly active antiretroviral therapy (HAART) became clinically available, about 60–80% of HIV-infected persons developed one or more episodes of Pneumocystis carinii pneumonia (PCP) unless given prophylaxis (Hughes et al., 1987Go; Masur et al., 1989Go). To prevent the occurrence and reoccurrence of PCP in HIV-infected persons, the Centers for Disease Control and Prevention (CDC) recommended that individuals with CD4 cell counts below 200/mm3 should receive prophylactic therapy (CDC, 1989) and later recommended the use of a combination of trimethoprim and sulfamethoxazole (TMP-SMX, 1:5) (CDC, 1992). When administered concomitantly, TMP (a dihydrofolate reductase inhibitor [Allegra et al., 1987Go]) and SMX (an inhibitor of dihydropteroate synthetase [Kovacs et al., 1989Go]) are efficacious for acute treatment and for prophylaxis of PCP (Hardy et al., 1992Go; Schneider et al., 1992Go). A high incidence (40–80%) of adverse reactions, including hypersensitivity reactions and hematological abnormalities, limited the clinical usefulness of TMP-SMX in HIV-infected patients receiving the antiretroviral drug zidovudine (ZDV, formerly known as azidothymidine, AZT) (Gordin et al., 1984Go; Kovacs et al., 1984Go; Medina et al., 1990Go; Wharton et al., 1986Go).

The adverse reactions associated with TMP-SMX therapy have been attributed to bioactivation of SMX resulting in formation of potentially toxic metabolites: hydroxylamine metabolite (SMX-HA), and nitroso metabolite (SMX-NO) (Cribb et al., 1991Go, 1996Go; Rieder et al., 1989Go; Shear et al., 1986Go). SMX-NO has been postulated to be the ultimate toxic species. Several studies have demonstrated that reactive metabolites of SMX cause toxicity either directly or by initiating immune-mediated reactions. For example, in vitro studies by Carr et al. (1993) demonstrated that SMX-HA is cytotoxic for lymphocytes of patients with a clinical history of hypersensitivity reactions to SMX. Naisbitt et al. (1999) also showed that peripheral blood lymphocytes of healthy individuals are sensitive to direct toxic effects of both SMX-HA and SMX-NO but not to the parent compound. The finding that addition of glutathione (a hydroxylamine scavenger) to lymphocyte cultures reduced the cytotoxic effects of SMX-HA suggests that glutathione deficiency, commonly encountered in HIV-seropositive patients (Buhl et al., 1989Go), contributes to the increased occurrence of adverse reactions in patients with AIDS. These results are consistent with the observation that in immunosuppressed HIV-seronegative patients receiving TMP-SMX therapy, the rate of adverse reactions is only 15% (Gordin et al., 1984Go; Winston et al., 1980Go). Patients with AIDS as well as HIV-seronegative persons treated with high-dose TMP-SMX have circulating SMX-specific IgG and IgM antibodies (Daftarian et al., 1995Go) and display CD4 and CD8 T-cell responses to SMX-HA (van der Ven et al., 1991Go; von Greyerz et al., 1999Go). When applied in vitro, SMX-HA was immunosuppressive as well. For example, SMX-HA inhibited natural killer (NK) cell activity (Leeder et al., 1991Go) and the phytohemagglutinin (PHA)-induced proliferative response of peripheral blood mononuclear cells without having a significant effect on cell viability (Hess et al., 1997Go; Rieder et al., 1992Go).

To determine whether interaction with ZDV increases the immunotoxic effect of TMP-SMX, we examined the effects of subchronic (28 days) oral administration of these anti-Pneumocystis carinii drugs alone or in combination with the anti-retroviral drug zidovudine (ZDV, formerly known as azidothymidine, AZT), on the cellular immune response of BALB/c mice by using a battery of assays modeled after the guidelines of the National Toxicology Program (Luster et al., 1988Go). We also examined the effect of these drugs on several hematological parameters, because normal hematopoiesis is essential for maintaining the immune system. We considered this study germane because the metabolic profile of SMX in rodents (Gill et al., 1997Go) is similar to that reported in humans (Vree et al., 1995Go). Moreover, the inherent myelosuppression from HIV, the presence of underlying infections (e.g., P. carinii), a deficiency in systemic glutathione (Buhl et al., 1989Go), and genetic differences in detoxification of oxidative metabolites of sulfonamides (Rieder et al., 1991Go; Shear et al., 1986Go) make it difficult to discern the effect of combined drug therapy in the clinical setting.

Our study showed that subchronic oral administration of TMP-SMX alone had no effect on hematopoiesis or cellular immune function in mice. The only evidence of hematotoxicity in mice treated with ZDV alone was the occurrence of mild macrocytic anemia, and no adverse immunological effects were discerned in mice treated with ZDV alone. However, when these drugs were given in combination, TMP-SMX synergized with ZDV, causing severe anemia, thrombocytopenia, lymphopenia, and neutropenia. Combined administration of TMP-SMX and ZDV also resulted in a marked drop in splenic cellularity, a significant decrease in the proportion of splenic macrophages, and suppression of accessory cell (AC)-dependent T-cell responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
ZDV (3`-azido-3-deoxythymidine) was provided by the Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID). Trimethoprim (2,4-diamino-5-[3,4,5-trimethoxybenzyl]pyrimidine), sulfamethoxazole (4-amino-N-[5-methyl-3-isoxazolyl]benzenesulfonamide), methylcellulose (MC), and concanavalin A (ConA) were obtained from Sigma (St. Louis, MO). Lipopolysaccharide (LPS) was purchased from List Biologics (Sunnyvale, CA). RPMI 1640 with 25 mM HEPES was obtained from BioWhittaker (Walkersville, MD). Iscove's modified Dulbecco's medium (IMDM), fetal calf serum (FCS), L-glutamine, penicillin, streptomycin, 2-mercaptoethanol (2-ME), streptavidin RED670, and monoclonal antibodies (mAb) to CD3, CD4, and CD8 were obtained from Life Technologies, Inc. (Grand Island, NY). Fluorescein isothiocyanate (FITC)-conjugated mAb to MAC-1 (a macrophage marker) and phycoerythrin (PE)-conjugated mAb to CD45R/B220 (a B cell marker) were obtained from Pharmingen (San Diego, CA). Methyl-[3H]thymidine (6.7 Ci/mmol) was purchased from NEN Life Sciences Products (Boston, MA). Methoxyflurane was purchased from Pitman Moore (Mundelein, IL).

Animals.
Five- to six-week-old female BALB/c mice were obtained from the Jackson Laboratories (Bar Harbor, ME) and quarantined for 7 days before initiation of the study. For each experiment, 3 animals were randomly selected for a quarantine necropsy, which included examination of all tissues and determination of tissue weights of the brain, kidneys, liver, and spleen. Feces were examined for endo- and ectoparasites, and blood was collected and submitted for serological evaluation of bacterial and viral infections. The results of these examinations and the general appearance of the animals were evaluated by the attending veterinarian, and animals were judged to be healthy and free from disease and parasites before release from quarantine. Mice were housed 3 per cage in polycarbonate cages. Animal rooms were environmentally controlled with a 12-h light/dark photocycle. All animals had food and water freely available. This research was approved by the Animal Studies Committee at SRI International and conformed to Society of Toxicology guidelines for experiments using animals.

Cell line.
The murine EL-4 cell line, a lymphoma from a C57BL/6N mouse, was obtained from the American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 with 8% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Preparation and analysis of dose preparations.
The TMP-SMX mixture was stable for less than one day and therefore was prepared daily and used within 1 h after preparation. This suspension was prepared by weighing out the appropriate amount of each test article, combining the dry powders, and dispersing them into 0.5% MC with the use of an Ultra Turrex homogenizer. Suspensions of ZDV were prepared weekly in 0.5% MC; when stored under refrigeration, this formulation was stable for at least 29 days. The estimated purity of the as-received test articles (TMP, SMX, and ZDV) was >98% compared to their USP standards.

The dose formulations were analyzed to verify dose concentration and homogeneity. For the TMP-SMX formulation, the ratio (1:5) and the concentration of both components were verified daily.

Similar analytical methods were used to analyze both the ZDV and the TMP-SMX formulations. ZDV and TMP-SMX were quantitated by high-performance liquid chromatography (HPLC) using a Waters Model 510 pump, a Bio-Rad Model AS-100 injector at 8°C, and a Waters Model 481 UV detector. The mobile phase was 20% acetonitrile/80% buffer at a flow rate of 1.0 ml/min. The buffer was prepared by mixing 500 ml water with 4 ml concentrated phosphoric acid and 1 ml triethylamine and adjusting to pH 6.2 by addition of solid potassium hydroxide.

Aliquots of ZDV dose suspensions were diluted in methanol and further diluted in water, and 25 µl injections of the samples were chromatographed on a reverse-phase C-18 column (Beckman-Altex, C-18, 4.6 x 250 mm) and detected at 267 nm. The retention time of ZDV was approximately 4.6 min. TMP-SMX formulations were sampled and the samples diluted in methanol, further diluted with mobile phase, and then chromatographed on a reverse-phase column (Regis, ODSII, 4.6 x 250 mm) and detected at 254 nm. The retention times of SMX and TMP were approximately 4.6 and 6.2 min, respectively.

Dose formulations within ±10% of the expected concentration were considered acceptable for use. In order to maintain homogeneity and prevent clogging of the needle, test articles were stirred continuously during dose administration.

Experimental design and sample collection.
To determine the effect of drug treatment on cellular immune function and hematopoiesis, 6 mice per treatment group received either TMP-SMX or ZDV alone, as a single oral daily dose in MC. Ten mice per group were given a combination of the 2 treatments, with the ZDV administered 30 min after the TMP-SMX. The treatment continued for 28 consecutive days. Control animals received MC alone. TMP-SMX was used at 1000 mg/kg (~160 mg/kg TMP and 840 mg/kg SMX) and ZDV at 240 mg/kg, because preliminary dose selection experiments showed 1500 mg/kg TMP-SMX and 480 mg/kg ZDV to be toxic. These groups are referred to hereafter as TMP-SMX, ZDV, TMP-SMX + ZDV, and MC, respectively. Mice were bled on Day 25 for hematological evaluation and euthanized one day after the last drug treatment (Day 29) for examination of tissues, spleen cell phenotyping, and determination of the splenocyte proliferative response ex vivo.

Animals were assigned to treatment groups on Day –1 by a computerized body weight stratification procedure and individually identified by a consecutively numbered earpunch. The volume administered was calculated for each mouse on the basis of the most recent body weight recorded by the LabcatTM data-capture software (Innovative Programming Associates, Inc., Princeton, NJ). Single daily doses were administered from Day 1 through Day 28. The dosing volume was 10 ml/kg. Animals were monitored once daily for morbidity (behavior, appetite, elimination, and clinical signs of ill health) and twice daily for mortality. Individual body weights were determined before initiation of treatment (Day –1) and weekly thereafter.

Hematological analysis.
Blood samples collected from the retro-orbital sinus into EDTA-coated tubes under methoxyflurane anesthesia were analyzed by IDEXX Veterinary Services, Inc. (West Sacramento, CA). Parameters measured included red blood cell (RBC) count, hemoglobin (HGB) concentration, hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLC), white blood cell (WBC) count, and WBC differential count, including lymphocytes (LYM) and segmented neutrophil (ANS).

Spleen cell isolation.
Spleens were weighed and cell suspensions were prepared using a Dounce homogenizer in media containing RPMI-1640 with 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Red blood cells were lysed using a solution containing 0.16 M ammonium chloride, 0.01 M potassium bicarbonate, and 0.096 mM EDTA. Spleen cell viability was determined by trypan blue exclusion.

Lymphocyte phenotyping.
Spleen cells were phenotyped by flow cytometry (FCM) using a Coulter Epics Elite Flow Cytometer (Hialeah, FL) and a previously described procedure (Freund et al., 1998Go). T cells were analyzed by 3-color FCM using FITC-conjugated mAb to CD3, biotin-conjugated mAb to CD4, and PE-conjugated mAb to CD8. Staining of biotin-conjugated CD4+ cells was developed using streptavidin RED670. The percentages of lymphocytes that express either CD4 or CD8 molecules were determined by gating on the CD3+ cell. B cells and macrophages were analyzed by 2-color FCM using PE-conjugated mAb to the CD45R/B220 molecule and FITC-conjugated mAb to MAC-1, respectively. Antibody to MAC-1 cross-reacts with granulocytes and NK cells as well. Ten thousand cells from each sample were analyzed using the Immuno-4 data analysis package (Coulter, Hialeah, FL).

Mitogen induced B and T cell proliferation.
Spleen cells (1 x 105 cells per well) were cultured in 200 µl of IMDM supplemented with 5% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5 x 10–5 M 2-ME (hereafter designated complete medium, CM). ConA or LPS (T cell and B cell mitogens, respectively) was added at a final concentration of 2.5 µg/ml or 10 µg/ml, respectively. The optimum concentration of each mitogen was determined in a preliminary experiment. Control cells were incubated in CM alone. All cultures were maintained for 72 h at 37°C in a humidified atmosphere containing 10% CO2. Cellular proliferation was measured by uptake of [3H]thymidine into the replicating DNA. Cultures were pulsed with 1 µCi [3H]thymidine per well 6 h before harvesting. The results from quadruplicate wells are expressed as the mean cpm ± SEM or as the stimulation index (SI), calculated as follows:

Mixed lymphocyte reaction (MLR).
The ability of T cells to proliferate in response to allogeneic cells was measured by incubating 1 x 105 spleen cells per well with 1 x 104 137Cs-irradiated (1500 rad) EL-4 cells in a total of 200 µl CM per well for 96 h under conditions described above. Control splenocytes were incubated in CM in the absence of stimulators. DNA synthesis in each culture was measured by uptake of [3H]thymidine during the last 16 h of incubation. The results from sextuplicate wells are expressed as the mean cpm ± SEM or as SI.

Statistical analysis.
Data are expressed as the mean ± SEM. The immune responses of various treatment groups were compared by Dunnett's test. Body and organ weights as well as hematologic parameters were compared by one-way analysis of variance (ANOVA) followed by Dunnett's test. Differences were considered significant at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clinical observations.
Oral administration of TMP-SMX, ZDV, or TMP-SMX + ZDV to mice did not result in death. Treatment with TMP-SMX + ZDV, but not other treatment regimens, resulted in weight loss; significant (p < 0.05) weight loss was initially observed on Day 7. These mice continued to lose weight; at termination of the study (Day 28), the mean body weight in the TMP-SMX + ZDV group was 18.6 ± 0.25 g, compared to 20.9 ± 0.39 g (p < 0.05) in the control group treated with MC (Table 1Go). Spleen weight in mice treated with TMP-SMX + ZDV was 63% lower than that in the MC group (p < 0.05) (Table 1Go). The mean spleen weight in the MC group was 116.6 ± 8.3 mg. No other gross abnormalities were noted at necropsy.


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TABLE 1 Effect of Oral Administration of Trimethoprim-Sulfamethoxazole and Zidovudine on Body and Spleen Weight and Spleen Cellularity
 
Hemotological profile.
Administration of TMP-SMX alone had no effect on hematological parameters, as determined by comparing these values to those for the MC group. Treatment with ZDV alone caused a low-grade macrocytic anemia evidenced by a significant (p < 0.01) decrease in RBC and HGB and a concomitant increase in MCV (52 ± 0.2 femtoliters [fl] vs. 47 ± 0.2 fl in the MC group; p < 0.01) (Table 2Go). The WBC and PLC remained unchanged in the ZDV group (Table 2Go). However, combined administration of TMP-SMX and ZDV led to severe pancytopenia, evidenced by significant (p < 0.01) anemia, thrombocytopenia, lymphopenia, and neutropenia (Table 2Go). The observed leukopenia in the TMP-SMX + ZDV group was primarily attributable to neutropenia; the ANS:LMY ratio in this group was 0.03, compared to 0.12 in the MC group.


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TABLE 2 Changes in Hematological Parameters in BALB/C Mice Treated Subchronically with a Combination of Trimethoprim-Sulfamethoxazole and Zidovudine
 
Cellular immune response.
The number of viable splenocytes (Table 1Go) and their subpopulation profile (Fig. 1A, 1B, and 1CGo) did not change following administration of either TMP-SMX or ZDV alone. The mean numbers of splenocytes in TMP-SMX and ZDV groups were 12.1 ± 1.1 x 107 and 13.4 ± 1.3 x 107, respectively, compared to 12.3 ± 0.9 x 107 in the MC group. However, TMP-SMX + ZDV produced a marked and statistically significant (p < 0.05) splenic hypocellularity (3.1 ± 0.7 x 107 cells/spleen) (Table 1Go), a 61% reduction (p < 0.05) in the percentage of macrophages (MAC-1+ cells) (Fig. 1AGo), and a concurrent 150% rise (p < 0.05) in the proportion of splenic CD3+ lymphocytes compared to values for MC-treated controls (Fig. 1BGo). The relative ratio of splenic CD4 and CD8 T-cell subsets (data not shown) and proportion of B cells (CD45R/B220+ cells) (Fig. 1CGo) were not altered by the combination treatment.



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FIG. 1. Effect of oral administration of TMP-SMX alone or in combination with ZDV on the cellular composition and ex vivo proliferative activity of murine splenocytes. Six mice per group were treated daily for 28 days with either the vehicle MC (clear bar), TMP-SMX (solid bar), ZDV (shaded bar), or TMP-SMX + ZDV (striped bar). One day after the last treatment, spleen cells were analyzed for the expression of cell surface molecules (MAC-1, CD3, and CD45R/B220) by flow cytometry (A–C, respectively) and proliferative responses to either ConA, EL-4 cells, or LPS (D–F, respectively) by uptake of [3H]thymidine into replicating DNA. The results are expressed as the mean ± SEM. *p < 0.05 when compared to MC group.

 
Oral administration of either TMP-SMX or ZDV alone had no effect on the ex vivo proliferative responses of splenic T or B lymphocytes (Fig. 1D, 1E, and 1FGo). The LPS-induced proliferative activity of splenic B cells from mice treated with TMP-SMX + ZDV was indistinguishable from that of the control MC-treated mice (Fig. 1FGo). TMP-SMX + ZDV did not impair the ability of T cells to respond to allogeneic cells (Fig. 1EGo). In fact, splenocytes from the TMP-SMX + ZDV group showed an 83% increase (p < 0.05) in DNA synthesis following stimulation with EL-4 cells (83,098 ± 3,567 cpm, vs. 45,444 ± 2059 cpm in the MC group; SI values 22 and 13, respectively). This finding is compatible with the observed 150% increase in the percentage of splenic CD3+ lymphocytes in the TMP-SMX + ZDV group (Fig. 1BGo). Consistent with the 61% reduction in the percentage of macrophages (Fig. 1AGo), spleen cells from the TMP-SMX + ZDV group showed 37% decrease (p < 0.05) in DNA synthesis when stimulated with the T-cell mitogen ConA (221,868 ± 15,128 cpm vs. 354,557 ± 11,609 cpm in the MC group; SI values 58 and 120, respectively) (Fig. 1DGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we investigated the effect of subchronic oral administration of TMP-SMX alone or in combination with ZDV on hematopoiesis and the cellular immune response of BALB/c mice. TMP and SMX were administered in a 1:5 ratio as in the commercial formulation of the drug. The daily doses of ~160 mg/kg TMP and ~840 mg/kg SMX used to treat mice were chosen based on body surface area (Chappel and Mordenti, 1991Go), to mimic the toxic effects observed when humans were treated daily with 15 to 20 mg/kg TMP and 75 to 100 mg/kg SMX as primary prophylaxis against PCP (Gordin et al., 1984Go; Safrin et al., 1996Go; Sattler et al., 1988Go). Mice often tolerate much larger doses of cytotoxic drugs (approximately 13 times higher on a body weight basis) than humans (Forsgren et al., 1980Go). Moreover, HIV-infected persons have a systemic glutathione deficiency that results in a decreased capacity to detoxify oxidatively generated SMX metabolites (Buhl et al., 1989Go). The ZDV dose used in this study was 240 mg/kg, a level that is known to cause macrocytic anemia in mice after long term oral treatment (Freund et al., submitted; Luster et al., 1989).

Our data demonstrate that treatment with TMP-SMX alone had no effect on hematopoiesis (Table 2Go) or cellular immune function (Figs. 1A–1FGo) of BALB/c mice. Our finding in mice confirms earlier observations by Viora et al. (1996), who showed that daily oral administration of 250 mg/kg TMP-SMX for 4 weeks had no effect on the ability of rat splenocytes to respond ex vivo to T-cell mitogens, ConA, or PHA. Anderson et al. (1980) also found that ingestion of four 480-mg TMP-SMX tablets (2 tablets twice daily) for 5 days did not affect the PHA- and ConA-induced proliferative activity of human T cells.

The disparity between the ex vivo (Anderson et al., 1980Go) and in vitro results (Hess et al., 1997Go; Naisbitt et al., 1999Go; Rieder et al., 1995Go) might be related to the short biological half-life of reactive SMX metabolites coupled with the quick recovery of immune cells from drug-mediated immunosuppression. In our ex vivo experiments, splenocytes were stimulated one day after the last drug administration. Plasma concentrations of SMX metabolites were not measured at the time of these assays. In rats, both SMX-HA and SMX-NO are extensively reduced to the parent compound and the inactive acetate (Gill et al., 1997Go), and a similar time course might be expected in mice. Reduction of oxidative metabolites of SMX to the parent drug has been reported with human liver microsomes (Cribb et al., 1995Go) as well.

Administration of ZDV alone caused a low-grade macrocytic anemia (Table 2Go) but did not alter the cellular composition or ex vivo proliferative activity of splenocytes (Figs. 1A–1FGo). Results of studies by Luster et al. (1989), Cronkite and Bullis (1990) and this laboratory (Freund et al., submitted) have demonstrated that mice develop macrocytic anemia after long-term treatment with ZDV. Use of ZDV in patients with AIDS and AIDS-related complex is associated with anemia as well (Medina et al., 1990Go; Richman et al., 1987Go; Sattler et al., 1988Go; Schneider et al., 1992Go). Our finding that administration of ZDV did not affect the ex vivo proliferative response of murine splenoctyes is in keeping with earlier observations by Luster et al., (1988), who showed that oral administration of 100–600 mg/kg ZDV for 30 days did not reduce the ability of spleen cells from C57BL/6 mice to respond to alloantigen ex vivo.

The hematological and immunological profiles of mice treated with TMP-SMX + ZDV differed markedly from those of mice treated with either TMP-SMX or ZDV alone. Co-administration of TMP-SMX + ZDV resulted in depletion of all cellular elements in the blood (Table 2Go), severe hypocellularity in the spleen (Table 1Go), a marked decrease in the percentage of splenic macrophages (Fig. 1AGo), and a concomitant reduction in ConA-induced T-cell proliferation (Fig. 1DGo), although the ability of splenic T cells to respond to allogeneic cells (EL-4 cells) was not affected (Fig. 1EGo).

For ConA to stimulate optimal proliferation of polyclonal T cells, it must induce the production of interleukin-2 (IL-2) and the expression of membrane receptors for IL-2 (IL-2R) (Larsson and Coutinho, 1980). Both ConA-induced IL-2 synthesis by unfractionated murine T cells (Larsson et al., 1980Go; Smith et al., 1980Go) and IL-2R expression by MHC class-II-restricted CD4+ T cells (Malek et al., 1985Go) require the participation of AC (accessory cells, e.g., macrophages). MHC class I-restricted CD8+ T cells express substantial levels of IL-2R in the absence of AC (Malek et al., 1985Go). Although the design of our study did not include AC reconstitution experiments, replenishment of splenic T cell cultures with drug-untreated syngeneic macrophages is expected to reconstitute ConA-induced T-cell proliferation in the TMP-SMX + ZDV group.

The increased incorporation of [3H]thymidine by splenocytes stimulated with EL-4 cells (Fig. 1EGo) is compatible with (1) the increased percentage of CD3+ cells in this organ (Fig. 1BGo), and (2) the ability of the T-cell lymphoma line, EL-4, to function as an AC. EL-4 cells are known to promote expression of IL-2R and to reconstitute the production of IL-2 as well as proliferation of CD4+ T cells (Farrar et al., 1980Go; Malek et al., 1985Go). Ia antigen recognition is not mandatory for EL-4 AC function, because Ia EL-4 cells were also able to function as efficient AC for induction of IL-2R expression in CD4+ T cells (Malek et al., 1985Go).

The rise in the relative proportion of CD3+ splenocytes in the TMP-SMX + ZDV group might be due to sequestration of T lymphocytes in the spleen. Activated T cells are preferentially sequestered in lymphoid tissue (Nakajima et al., 1994Go). SMX metabolites activate T cells without the need of uptake, metabolism, and processing by macrophages (Schnyder et al., 1997Go); T-cell activation results in increased expression of adhesion molecules known to mediate lymphocyte sequestration in lymphoid tissue (Nakajima et al., 1994Go). Phenotyping data on the matched peripheral blood samples and the frequency of activated splenocytes (H-2 Ia+ cells) expressing adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM-1) were not measured at the time the immune assays were conducted to verify these possibilities. Finally, the institution of TMP-SMX + ZDV treatment did not change the percentage splenic B cells (Fig. 1CGo) or their LPS-induced proliferative activity (Fig. 1FGo).

The occurrence of cytopenia in mice corroborates the results of clinical studies showing that, when used in conjunction with TMP-SMX, ZDV induces a higher incidence of anemia, thrombocytopenia, and severe neutropenia in patients with AIDS (Gordin et al., 1984Go; Kovacs et al., 1984Go; Medina et al., 1990Go; Richman et al., 1987Go; Wharton et al., 1986Go). A likely explanation for the differential extent of toxicity observed in neutrophils (Table 2Go) is the ability of neutrophils to accumulate SMX (Climax et al., 1986Go) and to produce cytotoxic intermediates intracellularly by peroxidase-dependent oxidation of this parent drug (Cribb et al., 1990Go). In the current study, the neutrophil:lymphocyte ratio in the TMP-SMX + ZDV group was 0.03, compared to a value of 0.12 in the control MC-treated group. Consistent with our findings, in vitro studies by Naisbitt et al. (1999) showed that both SMX-HA and SMX-NO, but not SMX, increased the rate of spontaneous apoptosis in human neutrophils; in contrast, lymphocytes were not affected. Intracellular formation of cytotoxic SMX metabolites could have contributed to the depletion of splenic macrophages (Fig. 1AGo). SMX is oxidized in monocytes and tissue macrophages as well (Cribb et al., 1990Go). Further work is required to determine the relative contribution of extrahepatic (tissue-specific) metabolism of SMX to clinical toxicity.

It is unlikely that the hematotoxic and immunotoxic effects observed in mice treated with TMP-SMX + ZDV were caused by elevated blood levels of ZDV or its main metabolite, because concomitant oral administration of TMP-SMX did not significantly alter the pharmacokinetic disposition of ZDV (Canas et al., 1996Go). ZDV has a short half-life (30–60 min) in mice (Pacifici et al., 1992Go). Although our interpretation is speculative, results from the current study in mice suggest that the observed hematological and immunological changes in mice resulted from synergistic interaction of ZDV and SMX metabolites; the majority of adverse reactions to TMP-SMX are mediated by the oxidative metabolites of SMX (Rieder et al., 1995Go; van der Ven et al., 1991Go). The higher incidence of hematological abnormalities (40–80%) in AIDS patients treated with TMP-SMX (Hughes et al., 1987Go; Sattler et al., 1988Go) than in non-HIV-infected immunocompromised patients (15%) (Gordin et al., 1984Go; Winston et al., 1980Go) and the observation that these abnormalities resolved when TMP-SMX therapy was discontinued (Gordin et al., 1984Go) support this assertion.

Further studies exploring the interactions of SMX and ZDV are needed because the issue of drug-induced bone marrow toxicity and the resulting deterioration of immune function is a concern accompanying the use of TMP-SMX in patients receiving ZDV. ZDV is a component of HAART therapy (Cameron et al., 1998Go; Hammer et al., 1997), and prophylaxis for PCP during the first 2 months of successful HAART therapy as well as maintenance therapy for previously diagnosed PCP has been recommended recently (Michelet et al., 1998Go). HAART is not without adverse side effects, and adherence to this complex drug regimen has proven difficult (Stephenson, 1998Go, 1999Go). Moreover, HAART is unaffordable for most individuals in developing countries (Arya, 1998Go).


    ACKNOWLEDGMENTS
 
This work was supported by NIAID Contracts NO1-AI-15111 and NO1-AI-65307. We thank Dr. Charles Litterst of NIAID for his scientific contributions to the design of these studies and critical review of the manuscript, Dr. Steven C. Miller for flow cytometric analysis, and Dr. Linda Werner of IDEXX Veterinary Services, Inc., West Sacramento, CA, for helpful discussions.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (650) 859-3153. E-mail: yvonne.freund{at}sri.com. Back

2 Present address: Office of Testing and Research, Food and Drug Administration, Rockville, MD 20857. Back


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