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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Transplant Proc. Author manuscript; available in PMC 2010 October 21.
Published in final edited form as:
Transplant Proc. 1991 December; 23(6): 2957–2958.
PMCID: PMC2958665
NIHMSID: NIHMS240818

Evaluation of the Influence of FK 506, Rapamycin, and Cyclosporine on Processing and Presentation of Particulate Antigen by Macrophages: Assessment of a Drug “Carry-Over” Effect

FK 506 is 10 to 100 times more potent than cyclosporine (CyA) in inhibiting T-cell responses to mitogenic and allostimulatory signals in vitro.1 Both drugs affect the early events subsequent to antigen–T-eell receptor interaction that characterizes T-cell activation.1 In contrast, rapamycin, which is structurally similar to FK 506, inhibits cytokine-mediated, but not T-cell receptor-mediated activation.1 The processing and presentation of antigen by macrophages is integral to the early events of T-cell activation. A recent report indicates that CyA interferes with these macrophage functions.2 In view of this observation, we examined the effects of FK 506 and rapamycin on the processing and presentation of particulate antigen using a model composed of bone marrow-derived macrophages, heat-killed Listeria monocytogenes (HKLM), and ? a listerial-reactive CD4+ T-cell clone.

MATERIALS AND METHODS

Animals

Eight- to 12-week-old female C57BL/6J mice purchased from Jackson Laboratories, Bar Harbor, Me, were used in all experiments. The animals were housed in accordance with the guidelines proposed by the Institute of Laboratory Animals Resources, National Research Council.

Bone Marrow-Derived Macrophages

Murine bone marrow-derived macrophages were generated in the presence of L-cell–conditioned medium as previously reported.3 Cells harvested from day 11 cultures were used for the experiments described.

Bacteria

The culture and maintenance of L monocytogenes (EGD strain) have been reported previously.4 HKLM was prepared by incubating bacterial suspensions for 1 hour at 60°C. HKLM was washed twice, suspended in saline at 1 × 109 cells/mL, and frozen at −80°C until required.

CD4+ T-Cell Clone

The production and characterization of the Literia-specific T-cell clone 5A9 have been previously described.5 Viable cells were purified by Ficoll-Hypaque centrifugation prior to use.

Immunosuppressants

FK 506 was a gift from Fujisawa Pharmaceutical Company Ltd, Osaka, Japan. Rapamycin was a gift from Wyeth-Ayerst Laboratories, Princeton, NJ. CyA was purchased from Sandoz Pharmaceuticals, Basel, Switzerland. All drugs were obtained in pure powder form. Ten millimolar stock solutions were prepared in absolute ethanol. All subsequent dilutions were made in tissue culture medium (RPMI-1640).

Experimental Assay of Antigen Processing and Presentation

Bone marrow-derived macrophages (3 × 104 cells/well) were seeded into 96-well microtiter plates and incubated for 2 hours at 37°C in order to allow the cells to attach. FK 506, rapamycin, or CyA was added and the cells were incubated for an additional 2 hours. The cell monolayer was then washed four times according to a protocol considered adequate for the removal of CyA.6 One million HKLM and 3 × 105 5A9 T lymphocytes suspended in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1 mmol/L sodium pyruvate, 1 mmol/L l-glutamine, 1% essential and nonessential amino acids (M.A. Bioproducts, Walkerville, Md), 5 × 10−5 M 2-mercaptoethanol, and 1% penicillin/streptomycin/fungizone (Gibco, Grand Island, NY) were added to each well and the cells were cocultured for 3 days. To assess T-cell proliferation, each well received 1 μCi of tritiated thymidine during the last 18 hours of the culture period and the counts incorporated were determined by liquid scintillation counting.

Concanavalin A Stimulation of Normal Splenocytes

Normal mouse splenocytes (1 × 105 cells per well) and 2 μg/mL of concanavalin A (ConA) (Sigma Chemicals, St Louis, Mo) were added to cultures of drug-treated macrophages. Splenocyte proliferation was assessed by tritiated thymidine incorporation on day 3.

RESULTS

FK 506, CyA, or rapamyein-pretreated macrophages suppress the proliferative response of 5A9 T cells to HKLM. The proliferative response of 5A9 T cells to HKLM was significantly reduced when the cells were cocultured with macrophages pretreated with drug (Fig 1). This inhibitory effect was dose-dependent. FK 506 was more potent than rapamycin which, in turn, was more potent than CyA.

Fig 1
Antigen-specific T-cell proliferation is suppressed in co-cultures containing macrophages pretreated with FK 506, CyA, or rapamycin. 5A9 T lymphocytes and HKLM were cocultured for 3 days with bone marrow-derived macrophages pretreated with drug at the ...

FK 506, CyA, and rapamyein-pretreated macrophages suppress the blastogenic response of normal splenocytes to ConA. Studies were undertaken to determine whether the inhibitory effects of these drugs on T-cell proliferation were due to drug carry over rather than on the capacity of macrophages to process and/or present antigen. Macrophages were incubated with various concentrations of each drug as above. The cell monolayer was then washed four times, and normal splenocytes and ConA (a stimulant that induces T-cell proliferation independently of our drug-treated macrophages of interest) were then added. T-cell proliferation was measured as previously. A significant suppression of the T-cell response to ConA was seen when cells were cocultured with macrophages pretreated with drug (Fig 2). This effect was much more pronounced with FK 506 than with rapamycin. In the case of CyA, appreciable impairment was only found in cocultures containing macrophages preexposed to 1,000 nm. These data demonstrate that the diminished proliferative response of 5A9 T lymphocytes to HKLM observed in cocultures containing macrophages pretreated with drug was mainly due to drug carry over rather than the effect of the drug on antigen processing and/or presentation.

Fig 2
ConA response of normal splenocytes is suppressed in cocultures containing drug-pretreated macrophages. Normal splenocytes and ConA (2 μg/mL) were cocultured with bone marrow-derived macrophages pretreated with drug at the concentrations indicated. ...

DISCUSSION

The incubation of HKLM and 5A9 T cells with drug-pretreated, macrophages resulted in significant suppression of antigen-induced T-cell proliferation. This suppression occurred regardless of whether macrophages were treated with FK 506, CyA, or rapamycin. The nonspecific proliferation of splenocytes to ConA was also inhibited in cocultures containing drug-treated macrophages indicating our inability to wash drug from out of our system.

Our results confirm previous studies by others in which a significant suppression of the T-cell response to antigen was observed when the cells were cocultured with macrophages pretreated with CyA.2 These latter studies, however, gave little credence to the possibility of drug carry over.

We are currently exploring alternative methods of washing drug-treated macrophages, in an effort to eliminate the direct effect of drug carry over on T-cell function. The development of such methodology would then enable us to assess the effects of immunosuppressants on the ability of macrophages to process or present antigen to T cells.

Acknowledgments

Supported by Grants DK 29961 (to T.E.S.) and AI 24141 (to E.J.W.), National Institutes of Health, Bethesda, Maryland.

REFERENCES

1. Dumont FJ, Staruch MJ, Koprak SL, et al. J Immunol. 1990;144:251. [PubMed]
2. Palay DA, Cluff CW, Wentworth PA, et al. J Immunol. 1986;136:4348. [PubMed]
3. Gregory SH. J Leukoc Biol. 1988;43:67. [PubMed]
4. Wing EJ, Waheed A, Shadduck RK. Infect Immun. 1984;45:180. [PMC free article] [PubMed]
5. Magee DM, Wing EJ. J Immunol. 1988;141:3203. [PubMed]
6. Hess AD, Tutschka PJ, Santos GW. J Immunol. 1981;126:961. [PubMed]