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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Transplantation. Author manuscript; available in PMC 2010 November 4.
Published in final edited form as:
Transplantation. 1994 February; 57(3): 433–439.
PMCID: PMC2972747
NIHMSID: NIHMS245183

RAPAMYCIN BUT NOT FK506 INHIBITS THE PROLIFERATION OF MONONUCLEAR PHAGOCYTES INDUCED BY COLONY-STIMULATING FACTORS1

Abstract

FK506, CsA, and rapamycin are potent inhibitors of T lymphocyte activation; relatively little is known of their effects on cells of the monocyte/macrophage lineage. Studies were undertaken to determine the effects of these drugs on the proliferative response of bone marrow-derived mononuclear phagocytes (BMMP) to CSFs. Rapamycin inhibited the proliferation of BMMP cultured in the presence of 10% L cell-conditioned medium, used as a source of macrophage CSF. The inhibition by rapamycin was dose dependent and apparent at concentrations of 0.1 nM or greater. In a similar fashion, rapamycin inhibited the proliferation of BMMP stimulated by the recombinant forms of murine IL-3 and murine granulocyte-macrophage CSF, and human macrophage CSF. In contrast, neither FK506 nor CsA at concentrations as high as 1000 nM diminished the proliferation of BMMP cultured under identical conditions. FK506, but not CsA, blocked the inhibitory effects of rapamycin on the response of BMMP to CSFs. In summary, these data indicate that rapamycin inhibits the proliferation of BMMP in response to CSFs. These results imply that patients receiving rapamycin, but not FK506 or CsA, may have an impaired ability to generate a functional mononuclear phagocyte population.

FK506 and rapamycin are structurally related, macrolide antibiotics derived from Streptomyces tsukubaensis (13) and Streptomyces hygroscopicus (4, 5), respectively. CsA, a cyclic peptide, is an unrelated fungal metabolite (6, 7). All 3 compounds exhibit potent immunosuppressive activity in vivo (811) and in vitro (1214). Although FK506 and CsA are structurally unrelated, they exhibit similar mechanisms of action. Both inhibit the early events in T cell activation that occur subsequent to antigen-TCR interaction and that culminate in the expression of early T cell activation genes (15). FK506 is 10–100 times more potent than CsA in suppressing the proliferation of mitogen- and alloantigen-activated T lymphocytes in vitro (12, 13). In addition, FK506 is more effective than CsA in suppressing graft rejection after organ transplantation in animal models (16). Both FK506 and CsA are used clinically to prevent graft rejection in humans (17, 18).

In contrast to either FK506 or CsA, rapamycin inhibits cytokine-driven T lymphocyte proliferation rather than the activation of cells induced by the antigen-TCR interaction (19). FK506 and rapamycin bind to the same intracellular protein, FK-binding protein (FKBP),* which is distinct from the intracellular binding site of CsA, cyclophilin (2022). As a consequence, FK506 and rapamycin act as reciprocal antagonists exerting mutually exclusive effects on T cell proliferation (13). Like FK506 and CsA, rapamycin effectively suppresses allograft rejection in animal models (23).

While the effects of rapamycin, FK506, and CsA on T lymphocytes are well documented, less is known of the effects of these drugs on other immune cell types, including mononuclear phagocytes. The proliferation and differentiation of bone marrow-derived mononuclear phagocytes (BMMP) from committed myeloid progenitors represent important features of the host's immune system (24). The growth factors responsible for the generation of a functional mononuclear phagocyte population constitute a family of glycoproteins termed the colony-stimulating factors. These factors include macrophage CSF (M-CSF), which stimulates the maximum proliferation of cells in this lineage, as well as granulocyte-macrophage CSF (GM-CSF) and IL-3. These CSFs may act alone or in combination to promote the proliferation and survival of BMMP in vivo and in vitro (24).

The present study was undertaken to investigate the effects of rapamycin, FK506, and CsA on the ability of BMMP to proliferate in response to CSFs. Here we report that CSF-driven BMMP proliferation is inhibited by pharmacological concentrations of rapamycin, but not of FK506 or CsA. These findings constitute one of the first reports to demonstrate that rapamycin differs from both FK506 and CsA in its effect on cells of the myeloid lineage. These data may have important implications with regard to the clinical use of rapamycin, FK506, and CsA in patients requiring immunosuppressive therapy, and to the mechanisms that underlie growth factor signaling in mononuclear phagocytes.

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. Animals were housed in accordance with the guidelines proposed by the Institute of Laboratory Animals Resources, National Research Council.

BMMP

BMMP were obtained as described previously (25, 26). Briefly, bone marrow was harvested from the femurs of mice killed by cervical dislocation. The cells were suspended in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sterile Systems, Inc., Logan, UT), 100 U/ml penicillin, 100 μg/ml streptomycin, 20% heat-inactivated equine serum (Sterile Systems) and 20% serum-free L 929 cell-conditioned medium (LCM), which served as a source of M-CSF. Bone marrow cells (2×105) in 20 ml of medium were seeded into 10-cm bacteriological grade polystyrene petri dishes, and the cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. On day 7 an additional 2 ml of LCM were added to all cultures. The cells were harvested after 11 or 12 days incubation, at which time they constituted a pure, relatively immature population of BMMP (25, 26).

CSFs

Recombinant human (rh) M-CSF and recombinant murine (rm) GM-CSF were the generous gifts of Immunex Corp., Seattle, WA; rmIL-3 was purchased from Genzyme Corp., Boston, MA. In preliminary experiments, 10,000 U/ml rhM-CSF, 1,000 U/ml rmGM-CSF, and 100 U/ml rmIL-3 stimulated the maximum proliferation of BMMP cultured under the conditions described.

Immunosuppressants

Rapamycin and FK506 were gifts obtained from Wyeth-Ayerst Laboratories, Princeton, NJ, and from Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan, respectively. CsA was obtained from Sandoz Pharmaceuticals, Basel, Switzerland. Stock 10-mM drug concentrations were prepared in absolute ethanol. Dilutions were made in RPMI 1640 medium supplemented with 10% FBS and antibiotics immediately before experimental use.

Assay of the effect of CSFs and drugs on the proliferation of BMMP

Microtiter plates were inoculated with BMMP suspended in RPMI medium supplemented with 10% FBS and antibiotics (5 × 103 cells/well) and the plates were incubated for 2 hr to allow the cells to attach. FK506, rapamycin, or CsA (ranging in concentration from 0.1 nM to 1000 nM) was then added with or without LCM, rhM-CSF rmGM-CSF, or rmIL-3 and cells were cultured for an additional period ranging from 4 hr to 7 days.

Assessment of cell proliferation

The proliferation of BMMP was assessed by one of the following 3 methods. (a) Metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): The ability of the BMMP to metabolize the tetrazolium salt MTT was used as a measure of cell proliferation (27). The cells were incubated with 5 mg/ml MTT (Sigma Chemical Co., St. Louis, MO) during the last 4 hr of the culture period. To solubilize the formazan product of MTT metabolism, an equal volume of 10% SDS in 0.1 N HCl was added to each well and the plates were incubated for an additional 18 hr at 37°C. The plates were read on a multiwell scanning spectrophotometer (ELISA reader) using a 570-nm test filter and a 630-nm reference filter.

(b) [3H]Thymidine incorporation: The cells were pulsed with 1 μCi/well [methyl-3H]thymidine (ICN, Costa Mesa, CA) during the last 18 hr of the culture period. The cells were collected using an automated cell harvester and [3H]thymidine incorporated into DNA was determined by liquid scintillation counting.

(c) Quantitation of cell number: The number of cells per well was assessed by lysing the cells with a 3% solution of hexadecyltrimethylammonium bromide (Fisher Scientific Co., Fair Lawn, NJ) and 1 mM EDTA in 0.85% saline, and counting the nuclei microscopically using a hemocytometer (28, 29).

Con A-induced splenocyte proliferation

Freshly isolated mouse splenocytes were suspended in RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium pyruvate, 1 mM L-glutamine, 1% essential and nonessential amino acids, 5 ×10-5 M 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were seeded into microtiter plates (1×105 cells/well) and incubated 3 days in the presence or absence of 1 μg/ml Con A and FK506, rapamycin, or CsA ranging in concentration from 0.1 to 1000 nM. [3H]Thymidine incorporation into DNA during the last 18 hr of the culture period was used as a measure of cell proliferation.

Statistical analysis

Means were compared using a nonpaired Student's t test; P<0.05 was statistically significant.

RESULTS

Rapamycin inhibits the proliferative response of BMMP to 10% LCM

Studies were performed to examine the effects of rapamycin, CsA, and FK506 on the kinetics of BMMP proliferation in response to 10% LCM, used as a source of M-CSF. Proliferation was assessed in terms of the ability of cells to metabolize the tetrazolium salt MTT. As shown in Figure 1, the inhibitory effect of 10 nM rapamycin on cell proliferation was evident on days 3, 5, and 7 of a 7-day incubation period. In contrast, neither FK506 nor CsA exerted an adverse effect on the proliferation of BMMP during this time. It is pertinent to note that none of the 3 immunosuppressants tested affected the degeneration or death of cells characteristic of BMMP cultured in the absence of an exogenous source of CSFs. Over the course of a 3-day incubation period, BMMP cultured in the absence of added CSFs exhibited a 5- to 10-fold loss in capacity to metabolize MMT regardless of whether FK506, CsA, or rapamycin was present at concentrations ranging from 0.001 to 1000 nM (data not shown).

Figure 1
Growth kinetics of BMMP cultured in the presence of CsA, FK506, or rapamycin. BMMP (5×103/6-mm well) were cultured in the presence of 10% LCM with or without 10 nM CsA, FK506, or rapamycin. Cell proliferation was assessed by the addition of MTT ...

Rapamycin inhibits the proliferation of BMMP in a dose-dependent fashion

Rapamycin, CsA, and FK506 were tested over a broad range of concentrations for their effects on the proliferative response of BMMP to 10% LCM. These effects were analyzed on the fifth day of incubation when the proliferation of cells peaked in control, untreated cultures. Rapamycin inhibited BMMP proliferation in response to LCM in a dose-dependent fashion (Fig. 2). Significant inhibitory effects were noted with 0.1 nM rapamycin; rapamycin at 10 nM or greater inhibited cell proliferation maximally. Neither FK506 nor CsA at concentrations ranging from 0.1 to 1000 nM affected the proliferation of cells cultured in the presence of 10% LCM.

Figure 2
Rapamycin inhibits BMMP proliferation in a dose-dependent fashion. BMMP (5×103/6-mm well) were incubated in the presence of 10% LCM and increasing concentrations of CsA, rapamycin, or FK506. On the fifth day of culture, MTT metabolism was used ...

Cell proliferation assessed in terms of MTT metabolism correlates with tritiated thymidine incorporation and cell number

To ensure that the results obtained by analysis of MTT metabolism were a true reflection of BMMP proliferation, cell number and [3H]thymidine incorporation into DNA were used in parallel experiments to assess proliferation. The addition of 10 nM rapamycin to BMMP cultured in the presence of 10% LCM resulted in a 50% decrease in MTT metabolism relative to untreated control cultures (Table 1). Similarly, cell cultures that contained rapamycin exhibited an approximate 75% decrease in both [3H]thymidine incorporation and cell number. Neither FK506 nor CsAhad an effect on BMMP proliferation as determined by each of the 3 assays.

TABLE 1
Rapamycin inhibits the proliferation of BMMP: correlation between MTT metabolism, [3H]thymidine incorporation, and cell numbera

Rapamycin inhibits the proliferative response of BMMP to the recombinant forms of human M-CSF, murine GM-CSF, and IL-3

The effects of rapamycin, CsA, and FK506 on the proliferative response of BMMP to recombinant CSFs correlated with their effects on the response of cells to LCM. Rapamycin at a concentration equal to or greater than 10 nM inhibited the proliferation of BMMP cultured in the presence of rhM-CSF (Fig. 3A); rapamycin (≥ 0.1 nM) inhibited the proliferation of cells induced by rmIL-3 (Fig. 3B). In addition, 1000 nM rapamycin inhibited the proliferation of cells cultured in the presence of rmGM-CSF (Fig. 3C). In contrast, neither FK506 nor CsA at concentrations ranging from 0.1 to 1000 nM adversely affected the proliferative response of cells to rhM-CSF, rmGM-CSF, or rmIL-3. In fact, the addition of 1000 nM FK506 appeared to increase the growth of BMMP cultured in the presence of rhM-CSF and rmIL-3. CsA at 1000 nM had a similar effect on the growth of cells cultured in the presence of rmIL-3.

Figure 3
Rapamycin inhibits the proliferative response of BMMP to rhM-CSF, rmGM-CSF, and rmIL-3. BMMP (5×103 cells/well) were incubated in the presence of (A) 10,000 U/ml rhM-CSF, (B) 100 Ulml rmIL-3, or (C) 1000 U/ml rmGM-CSF and increasing concentrations ...

Rapamycin, FK506, and CsA inhibit the proliferative response of splenocytes to Can A

Experiments were performed to compare the effects of rapamycin, FK506, and CsA on mitogen-stimulated splenocyte proliferation to their effects on BMMP proliferation. Normal mouse splenocytes were cultured in the presence of 1 μg/ml Con A and increasing concentrations of drug. Splenocytes cultured with either FK506 or rapamycin at concentrations equal to or greater than 0.1 nM and 0.001 nM, respectively, exhibited a marked reduction in cell proliferation relative to cells cultured in the absence of drugs (Fig. 4). Significant inhibition by CsA was observed at concentrations of 10 nM and greater. Thus, FK506 and CsA at pharmacological concentrations that had no effect on the proliferation of BMMP strongly suppressed the blastogenic response of splenocytes to Con A. In contrast, rapamycin at comparable concentrations inhibited the proliferation of both BMMP and splenocytes.

Figure 4
Rapamycin, FK506, and CsA inhibit the proliferation of Con A-stimulated splenocytes. Splenocytes (1 × 105/6-mm well) were cultured for 3 days in the presence or absence of 1 μg/ml Con A and increasing concentrations of CsA, FK506, or rapamycin. ...

FK506, but not CsA, antagonizes the inhibitory effects of rapamycin on BMMP proliferation

FK506, but not CsA, antagonizes the inhibitory effect of rapamycin on the proliferative response of T lymphocytes to selected stimuli, i.e., IL-2 and PMA (13). Experiments were undertaken, therefore, to determine whether FK506 also antagonized the effect of rapamycin on the proliferative response of BMMP to CSFs. As shown in Figure 5, rapamycin inhibited the proliferation of cells cultured in the presence of 10% LCM; FK506 and CsA had no effect. The inhibitory effect of rapamycin on BMMP proliferation was prevented by the addition of an equimolar concentration of FK506. CsA failed to prevent the inhibitory effect of rapamycin on BMMP proliferation.

Figure 5
FK506 reverses the inhibitory effect of rapamycin on BMMP proliferation. BMMP (5×103/well) were cultured in the presence of 10% LCM with or without CsA, FK506, or rapamycin (10 nM final concentrations) added singularly or in the combinations shown. ...

DISCUSSION

Previous studies have given little attention to the effects of immunosuppressants on the response of myeloid cells to CSFs. In the present study, pharmacological concentrations of rapamycin inhibited the proliferation of murine BMMP cultured in the presence of LCM, a source of M-CSF. Inhibition was evident regardless of the method used to assess cell growth, i.e., MTT metabolism, [3H]thymidine incorporation, or quantitation of cell number. The effect of rapamycin was dose dependent and apparent at concentrations comparable to those that inhibited the blastogenic response of mouse splenocytes to Con A. Similarly, rapamycin blocked the proliferation of BMMP cultured in the presence of rhM-CSF, rmGM-CSF, and rmIL-3. In contrast, FK506 and CsA failed to inhibit the proliferation of BMMP at drug concentrations that inhibited Con A-induced blastogenesis of splenocytes. None of the 3 immunosuppressants tested prohibited the death of BMMP cultured in the absence of CSFs. This latter finding suggests that the inhibitory effect of rapamycin on the proliferation of BMMP was exerted specifically on the response of cells to CSFs.

These findings correlate with published reports demonstrating that different mechanisms underlie the effects of FK506 and CsA compared with rapamycin on the activation or proliferation of T lymphocytes (12, 13). FK506 and CsA inhibit the early, calcium-dependent events in T cell activation that occur subsequent to antigen-TCR interactions (30). These early events are distinguished by the accumulation of intracellular calcium leading to the elevated expression of early phase activation genes which encode growth-promoting cytokines such as IL-2 and IL-4 (15). While both FK506 and CsA inhibit the production of IL-2 and IL-4 by T lymphocytes, neither impairs the calcium-independent proliferative response of cells to such cytokines (13). Rapamycin, on the other hand, inhibits the response of T cells to IL-2 and IL-4 without blocking cytokine production. Thus, it has been suggested that complexes formed between rapamycin and its cytosolic receptor preferentially block the signal transduction pathways used by growth factors (19).

FK506, CsA, and rapamycin exert effects on other immune cells that are similar to their effects on T lymphocytes. For example, FK506 and CsA inhibit the antigen receptor-mediated, calcium-dependent activation of B lymphocytes, but not the calcium-independent proliferative response of B lymphocytes to mitogens such as LPS or 8-mercaptoguanosine (31, 32). Similarly, FK506 and CsA inhibit the IgE receptor-mediated exocytosis of pharmacological mediators by human mast cells (3335). Rapamycin, on the other hand, blocks the mitogenic response of B lymphocytes to LPS and 8-mercaptoguanosine, and has no effect on receptor-mediated exocytosis exhibited by mast cells.

The contrasting effects of FK506 and rapamycin on cell activation are somewhat surprising in view of their structural similarity and their apparent affinity for the same intracellular binding protein, FKBP (30). The affinity of FK506 and rapamycin for the same cytosolic receptor was first suggested by studies demonstrating their capacity to act as reciprocal antagonists during the activation of murine T lymphocytes (13). It was later found that rapamycin readily displaced FK506 from FKBP in a competitive binding assay (36). More recent studies suggested that FK506 and rapamycm also compete for a common intracellular receptor in B lymphocytes (31) and mast cells (34). Similarly, in the study reported here, FK506 reversed the inhibitory effect of rapamycin on the proliferative response of BMMP to CSFs, suggestmg that FK506 and rapamycin may compete for a common intracellular receptor in BMMP. CsA did not prevent rapamycin-mediated suppression of BMMP proliferation. This is consistent with its interaction with an entirely different intracellular binding protein, cyclophilin (21, 22).

Further studies have helped to clarify the mechanisms that underlie the immunosuppressive effects of FK506, rap amycin, and CsA, and to resolve the different effects of FK506 and rapamycin on T cell activation. It is now believed that the biological activities of these immunosuppressants are mediated by complexes formed hetween the drugs and their intracellular binding proteins. For CsA and FK506, these complexes (CsA-cyclophilin and FK506-FKBP, respectively) bind and inactivate calcineurin, a protein phosphatase that plays a critical role in the early, calcium-dependent events that culminate in T cell activation (37, 38). In this regard, it is relevant to note that the mitogenic response of BMMP to the CSFs occurs in the apparent absence of calcium involvement (39). Thus, the failure of FK506 and CsA to suppress the proliferative response of BMMP to the CSFs implies that calcineurin is not a critical component of the activation pathway. While rapamycin binds to the same cytosolic receptor as FK506, available data suggest that the rapamycin-FKBP complex interacts with a molecule(s) other than calcineurin affecting the activation of T lymphocytes by calcium-independent mechanisms, e.g., those that mediate the response of cells to cytokines such as IL-2 and IL-4 (38). Our findings indicate that the proliferative response of BMMP to CSFs, similar to the response of T lymphocytes to IL-2 or IL-4, is mediated by a Ca++/calcineurin-independent pathway.

The findings reported here have important implications in view of the potential use of rapamycin, FK506, and CsA in a general clinical setting. Based upon experimental data demonstrating the efficacy of FK506 in preventing organ allograft rejection (11), autoimmune diseases (8), and graft-versus-host disease after allogenic BMT (40), clinical trials involving FK506 are proceeding in the United States and Europe. Clinical use of rapamycin is presently restricted to dose-response trials conducted only in certain centers. Since the proliferative response of BMMP to CSFs is an integral feature of hematopoiesis and host defenses (24), it is more likely that patients receiving rapamycin would be compromised in their abilities to generate a functional mononuclear phagocyte population and to respond to infection. On the other hand, the capacity of rapamycin to suppress BMMP proliferation could be used in the treatment of certain diseases, e.g., granulomatous diseases such as histiocytosis or leprosy, in which BMMP play an integral role in the pathogenesis. Thus, careful consideration given to the potent inhibitory effects of rapamycin on BMMP proliferation should allow rapamycin to be used in an efficacious manner.

Acknowledgments

The authors thank Dr. Susan A. McCarthy for her critical review of the manuscript.

Footnotes

1This work was supported by National Institutes of Health Grants DK 29961 (T.E.S.) and AI 24141 (E.J.W.).

*Abbreviations: BMMP, bone marrow-derived mononuclear phagocyte; FBS, fetal bovine serum; FKBP, FK-binding protein; GM-CSF, granulocyte-macrophage colony-stimulating factor; LCM, L cell-conditioned medium; M-CSF, macrophage colony-stimulating factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; rh, recombinant human; rm, recombinant murine.

REFERENCES

1. Tanaka H, Kuroda A, Marusawa H, et al. Structure of FK506: a novel immunosuppressant isolated from Streptomyces. J Am Chern Soc. 1987;109:5031.
2. Kino T, Hatanaka H, Hashimoto M, et al. FK 506, a novel immunosuppressant isolated from a streptomyces. 1. Fermentation, isolation and physico-chemical and biological characteristics. J Antibiot. 1987;60:1249. [PubMed]
3. Kino T, Hatanaka H, Susumu M, et al. FK-506, a novel immunosuppressant isolated from a streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J Antibiot. 1987;60:1256. [PubMed]
4. Vezina C, Kudelski A, Sehgal SN. Rapamycin (AY-22,989), a new fungal antibiotic. 1. Taxonomy of the producing streptomycete and isolation of the active principle. J Antibiot. 1975;28:721. [PubMed]
5. Sehgal SN, Baker H, Vezina C. Rapamycin (AY-22,989), a new fungal antibiotic. II. Fermentation, isolation and characterization. J Antibiot. 1975;28:721. [PubMed]
6. Borel JF, Feurer C, Magnee C, Stahelin H. Effects of the new anti-lymphocytic peptide cyclosporin A in animals. Immunology. 1977;32:1017. [PubMed]
7. Borel JF, Feurer C, Gubler HU, Stahelin H. Biological effects of cyclosporin A: a new anti-lymphocytic agent. Agents Actions. 1976;6:468. [PubMed]
8. Inamura N, Hashimoto M, Nakahara K, Aoki H, Yamaguchi I, Kohsaka M. Immunosuppressive effects of FK 506 on collagen-induced arthritis in rats. Clin Immunol Immunopathol. 1988;46:82. [PubMed]
9. Ochiai T, Sakamoto K, Gunji Y, et al. Effects of combination treatment with FK 506 and cyclosporine on survival time and vascular changes in renal-allograft-recipient dogs. Transplantation. 1989;48:193. [PubMed]
10. Todo S, Ueda Y, Demetris J, et al. Immunosuppression of canine, monkey and baboon allografts by FK 506 with special reference to synergism with other drugs and to tolerance induction. Surgery. 1988;104:239. [PMC free article] [PubMed]
11. Murase N, Kim DG, Todo S, Cramer DV, Fung JJ, Starzl TE. Suppression of allograft rejection with FK 506. 1. Prolonged cardiac and liver survival in rats following short-course therapy. Transplantation. 1990;50:186. [PMC free article] [PubMed]
12. Dumont FJ, Staruch MJ, Koprak SL, Melino MR, Sigal NH. Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK 506 and rapamycin. J Immunol. 1990;144:251. [PubMed]
13. Dumont FJ, Melino MR, Staruch MJ, Koprak SL, Fischer PA, Sigal NH. The immunosuppressive macrolides FK 506 and rapamycin act as reciprocal antagonists in murine T cells. J Immunol. 1990;144:1418. [PubMed]
14. Sawada S, Suzuki C, Kawase Y, Tanaka F. Novel immunosuppressive agent, FK 506: in vitro effects on cloned T cell activation. J Immunol. 1897;139:1797. [PubMed]
15. Tocci MJ, Matkovich DA, Collier KA, et al. The immunosuppressant FK 506 selectively inhibits expression of early T cell activation genes. J Immunol. 1989;143:718. [PubMed]
16. Ochiai T, Nakajima K, Nagata M, Hori S, Asano T, Isono K. Studies of the induction and maintenance of long term graft-acceptance by treatment with FK 506 in heterotopic cardiac allo-transplantation in rats. Transplantation. 1987;44:734. [PubMed]
17. Starzl TE, Todo S, Fung J, Demetris AJ, Venkataramman R, Jain A. FK 506 for liver, kidney and pancreas transplantation. Lancet. 1989;2:1000. [PMC free article] [PubMed]
18. Fung J, Abu-Elmagd K, Jain A, et al. A randomized trial of primary liver transplantation under immunosuppression with FK 506 vs cyclosporine. Transplant Proc. 1991;23:2977. [PMC free article] [PubMed]
19. Schreiber SL. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science. 1991;251:283. [PubMed]
20. Harding MW, Galat A, Uehling DE, Schreiber SL. A receptor for the immunosuppressant FK 506 is a cis-trans peptidyl-prolyl isomerase. Nature. 1989;341:758. [PubMed]
21. Siekierka JJ, Hung SHY, Poe M, Lin CS, Sigal NH. A cytosolic binding protein for the immunosuppressant FK 506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature (Lond) 1989;341:755. [PubMed]
22. Siekierka JJ, Staruch MJ, Hung SHY, Sigal NH. FK 506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporine A-binding protein, cyclophilin. J Immunol. 1989;143:1580. [PubMed]
23. Eng CP, Gullo-Brown J, Cheng JY, Sehgal SN. Inhibition of skin graft rejection in mice by rapamycin: a novel immunosuppressive macrolide. Transplant Proc. 1991;23:868. [PubMed]
24. Gregory SR, Magee DM, Wing EJ. The role of colony-stimulating factors in host defenses. Proc Soc Exp Biol Med. 1991;197:349. [PubMed]
25. Gregory SH. Substratum-dependent proliferation and survival of bone marrow-derived mononuclear phagocytes. J Leukocyte Biol. 1988;43:67. [PubMed]
26. Gregory SH. The viability of mononuclear phagocytes in vitro is diminished by the interaction of cells with serum proteins bound to the culture substratum. Immunol Lett. 1989;20:155. [PubMed]
27. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55. [PubMed]
28. Stewart CC, Yen S, Senior RM. Colony-forming ability of mononuclear phagocytes. In: Herscowitz HB, Holden HT, Bellanti JA, Ghaffar A, editors. Manual of macrophage methodology: collection, characterization and function. Marcel Dekker; New York: 1981. p. 171.
29. Yen S, Stewart CC. Effects of serum fractions on the growth of mononuclear phagocytes. J Cell Physiol. 1982;112:107. [PubMed]
30. Schreiber SL, Crabtree GR. The mechanism of action of cyclosporin A and FK506. Immunol Today. 1992;13:136. [PubMed]
31. Wicker LS, Boltz RC, Jr, Matt V, Nichols EA, Peterson LB, Sigal NH. Suppression of B cell activation by cyclosporin A, FK506 and rapamycin. Eur J Immunol. 1990;20:2277. [PubMed]
32. Kay JE, Kromwel L, Doe EA, Denyer M. Inhibition of T and B lymphocyte proliferation by rapamycin. Immunology. 1991;72:544. [PubMed]
33. De Paulis A, Cirillo R, Ciccarelli A, de Crescenzo G, Oriente A, Marone G. Characterization of the anti-inflammatory effect of FK-506 on human mast cells. J Immunol. 1991;147:4278. [PubMed]
34. Hultsch T, Albers MW, Schreiber SL, Hohman RJ. Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. Proc Natl Acad Sci USA. 1991;88:6229. [PubMed]
35. De Paulis A, Cirillo R, Ciccarelli A, Condorelli M, Marone G. FK 506, a potent novel inhibitor of the release of proinflammatory mediators from human FcERI+ cells. J Immunol. 1991;146:2374. [PubMed]
36. Bierer BE, Mattila PS, Standaert RF, et al. Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc Natl Acad Sci USA. 1990;87:9231. [PubMed]
37. Liu J, Farmer JD, Jr, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilincyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807. [PubMed]
38. Screiber SL, Liu J, Albers MW, et al. Immunophilin-ligand complexes as probes of intracellular signaling pathways. Transplant Proc. 1991;23:2839. [PubMed]
39. Vairo G, Hamilton JA. Signalling through CSF receptors. Immunol Today. 1991;12:362. [PubMed]
40. Cooper MH, Markus PM, Cai X, Starzl TE, Fung JJ. Prolonged prevention of acute graft-versus-host-disease after allogeneic bone marrow transplantation by donor pre-treatment using FK 506. Transplant Proc. 1991;23:3238. [PMC free article] [PubMed]