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FK506, like cyclosporine, diminishes IL-2 receptor expression and inhibits IL-2 production by T lymphocytes (1). IL-2 plays a crucial role in the generation and function of the large granular lymphocytes, also called natural killer cells, which are cytolytic in vitro against various tumor cells, some microorganisms, and virally infected cells (2). Because posttransplantation neoplasia and viral sepsis are common complications after transplantation under immunosuppression, we investigated the effects of a 4–14-day course of 1.0 mg/kg/day intramuscular FK506 on NK cell function in F344 rats. This dose has been shown to prevent allograft rejection in MHC- and non-MHC–incompatible donor-recipient strain combinations (3). Animals were sacrificed after 4, 7, or 14 days of treatment, and fresh mononuclear spleen cells were isolated after centrifugation on Ficoll-Hypaque gradients and passage over nylon-wool columns to remove monocytes/macrophages and B cells. NK activity of the cell isolates was determined by lysis of NK-sensitive YAC-1 tumor targets in a 4-hr 51Cr release assay. Antibody-dependent cytotoxicity, which is another measure of NK activity, was measured by lysis of NK-resistant P815 tumor targets in the presence of rat anti-P815 serum. No differences were found in NK and ADCC activity after 4 and 7 days of treatment with FK506 (Table 1). After 14 days, there was a slight increase in NK (17–19%) and ADCC (22%) activity. Because FK506 suppresses in vitro mixed lymphocyte reaction responses (1), we also performed these NK assays on fresh spleen cells obtained from untreated rats and exposed in vitro to 0.06–250 ng concentrations of FK506. There was no effect of FK506 on fresh splenic NK cell activity over this wide dose range (Fig. 1).
The results of flow cytometry studies on splenic cell isolates were congruent with the NK assay results. The lack of effect of FK506 on NK cell activity correlated with the expression of NK-associated cell surface markers. No differences were noted in the expression of CD2 (present on NK cells and T cells) and 3.2.3. (NK cell–specific) after 4, 7, and 14 days of treatment (Table 2, data of day 14 not shown). The number of LGL in cytospin preparations was not changed after 4 days of FK506 treatment and only slightly reduced after 7 days. Asialo-GM1–positive cells (which include NK cells, subpopulations of macrophages, and subsets of T cells) were increased from 25±7 (% positive cells ±SD) on day 4 to 36±1 on day 7; all values were within normal range.
The effect of FK506 on T lymphocytes in the same rats also was investigated in order to verify that the dosage regimen was therapeutic. This was a positive control for the IL-2-dependent T cell mechanism. Fresh mononuclear spleen cells from the control and FK506-treated animals were cultured for 3 days at a concentration of 1×105 cells/well in the presence or absence of Con A (2 μg/ml) or recombinant IL-2 (103 units/ml) and were subsequently pulsed for 8 hr with [3H]thymidine. There was no difference in response to stimulation with IL-2 between treated and control animals at days 4, 7, and 14 of FK506 treatment (Fig. 2). However, the response to Con A stimulation showed 53% (day 4), 24% (day 7), and 43% (day 14) inhibition of proliferation (Fig. 2).
Thus, although IL-2 has also been shown to be important for the generation and function of NK cells (2), our experiments showed that FK506, which is known to inhibit IL-2 production and receptor expression did not suppress NK or ADCC activity with either in vitro or in vivo test systems. The lack of an inhibitory effect on NK cytotoxicity correlates well with findings in T cells, in which FK506 also does not interfere with the cytotoxic function of effector T cells.
Some information is available about the effect of other immunosuppressive agents on NK activity. While most investigators have found an inhibition of NK and ADCC activity during azathioprine therapy, there are conflicting reports about corticosteroids and CsA (4–6). Gui et al. (4) reported NK inhibition with CsA. However, Muller et al. (6) reported that ADCC activity was inhibited during corticosteroid therapy in human transplant recipients while NK activity was not. Combined treatment with CsA and corticosteroids in their patients suppressed both NK and ADCC activity, and even more acute inhibition was found when azathioprine was added as a third agent. Such discrepancies may reflect differences in assays, patient populations, and extent and type of diseases or species studied.
In conclusion, we have shown that NK and ADCC function of spleen cells after 4 and 7 days of in vivo treatment with FK506 was not affected, whereas a slight increase (19% for NK and 22% for ADCC activity) was noted on day 14 compared with control animals. The number of NK cells, as determined by surface marker phenotype and by morphological analysis of the number of LGL, was not different compared with spleen cells from control rats. These findings indicate that FK506 has little or no effect on NK cell numbers or function in the rat.
1This work was supported by Research Grants from the Veterans Administration and by Project Grant DK 29961 from the National Institutes of Health, Bethesda, MD.