Prednisolone inhibits LPS-induced bone marrow suppressor cell activity in vitro but not in vivo

International Immunopharmacology 3 (2003) 169 – 178 www.elsevier.com/locate/intimp

Prednisolone inhibits LPS-induced bone marrow suppressor cell activity in vitro but not in vivo Kristi A. Haskins, Scott M. Schlauder, James H. Holda * Department of Biology, The University of Akron, Akron, OH 44325-3908, USA Received 18 May 2001; received in revised form 3 August 2001; accepted 20 August 2001

Abstract Glucocorticoids are used clinically to treat a variety of inflammatory diseases including endotoxemia. We hypothesized that injecting mice with the steroid prednisolone (pred) would mitigate the enhanced bone marrow (BM) natural suppressor (NS) cell activity that occurs in mice after receiving an injection of lipopolysaccharide (LPS). In vitro, prednisolone blocked the ability of NS cells to produce the immunosuppressive molecule nitric oxide (NO) and also the ability to suppress T cell proliferation. Prednisolone acted both indirectly, by blocking synthesis of cytokines necessary for NS cell activation, and also directly on NS cells, by blocking production of NO. In vivo, variable results were obtained. Prednisolone at 20 Ag/gm did decrease NS activity when injected into normal mice. However, when mice were injected with both LPS and prednisolone (0.2 or 20 Ag/gm), a large increase in BM NS activity was observed. The increase was evident in both the ability of the BM cells to suppress T cell proliferation and to produce NO. The data show that, in vivo, the steroid prednisolone in conjunction with the inflammatory compound LPS act to enhance BM NS activity. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide; Endotoxin; Bone marrow; Natural suppressor cells; Prednisolone

1. Introduction Endotoxemia is the 13th leading cause of death in the United States, especially affecting the young and the elderly [1]. Clinical manifestations of endotoxemia can include abnormal body temper-

Abbreviations: NS, natural suppressor; NO, nitric oxide; BM, bone marrow; TCM, tissue culture media; pred, prednisolone; SpC, spleen cells; LPS, lipopolysaccharide; IFN-g, interferon gamma; NOS2, nitric oxide synthase. * Corresponding author. E-mail address: [email protected] (J.H. Holda).

ature, hypotension, tachycardia, disseminated intravascular coagulation, and potentially multiple organ dysfunction syndrome (MODS) [2,3]. Treatment usually centers on blocking the synthesis or action of pro-inflammatory cytokines [1]. One current form of treatment uses anti-inflammatory drugs, such as the steroid prednisolone, to block cytokine synthesis [1,4,5]. However, despite best efforts, little progress has been made in the treatment of endotoxemia [1]. Experimentally, various models have been developed to induce symptoms of endotoxemia. In some models, LPS is injected into experimental animals, along with different therapeutic agents to test their

1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 2 ) 0 0 1 4 3 - 1

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efficacy as a treatment for endotoxemia [1]. These experiments usually look at a short time frame of less than 20 h [6]. One consequence of injecting LPS into mice is the development of increased BM NS cell activity [7]. The increase in NS activity peaks at 48-h post injection, and then wanes. Suppression is mediated by an increase in NO production by the BM NS cells [7]. Cells of various lineages have been implicated as having NS activity [8 –10]. Recently, cells with NS activity have been found to have CD31 on their surface, a marker for immature neutrophils [11]. NS cells have been shown to suppress T cell proliferation, antibody production, and mixed lymphocyte reactions [12]. Suppression is induced by the immunosuppressive molecule NO, and has been shown to be dependent on the inducible form of nitric oxide synthase (NOS2) [11,13]. The generation and activation of NS cells, both in vivo and in vitro, has been shown to be dependent upon T cell signals [14,15]. Recently, Angulo et al. [14] have shown that two signals are needed for NS cell activation. IFN-g is required for NS cell activity while several factors may act as the second signal. These authors also demonstrated that NS cells express CD40, and may be activated by interacting with the CD40 ligand on T cells [14]. Previously, Rodriguez et al. [16] have shown that glucocorticoids could block the ability of BM NS cells to suppress T cell proliferation in vitro. This effect was reversed by the addition of IFN-g to the cultures. In this paper, we extend the results of Rodriguez et al. and examine the effect of the steroid prednisolone on the ability of BM NS cells to produce the immunosuppressive molecule NO in vitro. We also examine the effect of prednisolone on the ability of LPS to enhance BM NS cell activity in vivo.

2.2. Reagents Con A was obtained from Calbiochem (LaJolla, CA). Murine recombinant IFN-g was obtained from Genentech (San Francisco, CA); units used were as defined by Genentech. Tissue culture media (TCM) consisted of RPMI 1640 supplemented with 2 mM Lglutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin, 10 mM Hepes, 5% fetal calf serum (GIBCO, Grand Island, NY) and 5  10 5 M 2-mercaptoethanol (Sigma, St. Louis, MO). Prednisolone 21-sodium succinate (Sigma) was used for in vitro studies at 0.3, 3.0, and 30 AM final concentrations. For injections, prednisolone (Sigma) was used at 0.2 or 20 Ag/g. Lipopolysaccharide (LPS) from S. typhosa (lot 123F4014) phenol extracted and chromatographically purified was obtained from Sigma. Stock solutions were prepared by dissolving the LPS in pyrogen-free phosphate buffer saline (PBS) (Sigma) to a final concentration of 1 mg/ml. The stock was frozen at 70 jC until use. 2.3. Injections Mice received injections of LPS alone (0.5 Ag/g), prednisolone alone, or a combination of both prednisolone and LPS. Two doses of prednisolone were used in separate experiments, either 20 or 0.2 Ag/g. Prednisolone was administered in two injections; the first injection was given 1 h before LPS and the second 20 h after LPS. Control animals that did not receive LPS were injected with pyrogen-free PBS. For prednisolone injections, the steroid was initially dissolved in dimethylsulfoxide (Sigma) (approximately 10% of the final volume) and pyrogen-free PBS was used for the remainder of the solution. The injection of LPS is considered time zero. Bone marrow was removed for assay 48 h after receiving the injection of LPS.

2. Materials and methods

2.4. Cell suspensions

2.1. Animals

All cell suspensions were prepared in cold Hanks balanced salt solution (HBSS). Spleen cell (SpC) suspensions were prepared by gently pressing the spleen through a 100-mesh stainless steel screen. BM cells were obtained by flushing the cells from the femur and tibia as previously described [7]. Red

Female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) 8 to 16 weeks of age were used in all experiments. All animals were treated in accordance with University IACUC policies.

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blood cells were removed from all cell suspensions by treatment with tris-buffered ammonium chloride. Cells were washed three times with HBSS and then adjusted to the appropriate concentration in TCM.

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diately or frozen at 70 jC. Typically, 48-h supernatants were frozen at 70 jC and then assayed at the same time as the 72-h supernatants. Preliminary tests indicated that freezing does not diminish detectable NO in the supernatants.

2.5. Enrichment of NS cells 2.8. Assaying for NO Plastic adherent cells were removed from all BM cell populations before use. To remove adherent cells, BM cells were adjusted to 5  106 cells per ml, in TCM, and then incubated on plastic for 1 to 2 h at 37 jC, 6% CO2. After incubation, the nonadherent cells were gently poured off and collected. 2.6. Suppression assay The ability of BM cells to suppress proliferation was determined by titrating BM cells into wells containing 2.5  105 SpC plus Con A at 2.5 Ag/ml; the final volume of the well was adjusted to 250 Al with TCM. Assays were performed in 96 well plates (Falcon, Lincoln Park, NJ); the cultures were incubated in a CO2 (6% CO2) incubator at 37 jC for 72 h. 1 ACi of 3H-thymidine (ICN, Costa Mesa, CA) was added for the last 18 h. After 72 h, the cultures were harvested onto glass filter strips using a MASH harvester and the amount of 3H-thymidine uptake determined using a Beckman liquid scintillation counter. To some cultures, prednisolone was added at the final concentrations mentioned previously. Cell density controls were run using normal spleen cells added to the cultures, at the same concentrations as those of the suppressors. 2.7. In vitro generation of NO NO production from normal and LPS BM was induced in two ways. First, 1.5  106 BM cells were placed into 24-well plates (Falcon) with 106 SpC and Con A (2.5 Ag/ml) in a final volume of 1 ml. In the second method, 1.5  106 BM cells were incubated with LPS and IFN-g. Preliminary experiments indicated that LPS at 1 Ag/ml and IFN-g at 5 units/ml were optimal (data not shown). Again, the final volume was 1 ml. In both cases, cultures were incubated for 48 or 72 h and supernatants assayed for NO activity. Supernatants to be tested were centrifuged 200  g for 10 min, and then assayed imme-

NO production was determined using the Greiss assay, which reduces NO to nitrite which, is then detected spectrophotometrically [17]. For each experiment, NO standards were made in TCM using NaNO2 (Sigma). A standard curve was run with each experiment, using 100, 50, 25, 12.5, and 6.25 AM NaNO2. Samples were added in 50-Al volumes to a 96-well plate, along with 50 Al of fresh Greiss reagent. The plate was incubated at room temperature for 5 min and colorimetric analyses were performed using a Dynatech MR 5000 plate reader. All assays were run in triplicate. For each experiment, a well containing TCM alone was run, typically giving an O.D. reading of 0.054 F 0.004. 2.9. Statistics All experiments were carried out at least twice. All proliferation assays were run in triplicate or quadruplicate, the standard deviation was usually less than 10% of the mean. Each sample for NO analysis was run in triplicate. To determine the concentration of NO contained in a sample, the statistical program Corel Quattro Pro was used. A linear regression was performed using the standards, to determine the concentration of the unknown sample. A statistical difference between samples was determined using a standard Student’s t-test.

3. Results Previously, Rodriguez et al. [16] showed that glucocorticoids blocked the ability of NS cells to suppress T cell proliferation. Table 1 shows similar results using the steroid, prednisolone. BM cells at 2.5  105 cells per well suppress Con A-induced proliferation of SpC by 86% (14% control). When prednisolone was added to the culture, suppression decreased. For example, when 0.3 AM prednisolone

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Table 1 Prednisolone partially removes the ability of BM NS cells to suppress Con A-induced proliferation in vitro Prednisolone (AM)

CPM BM 2.5  105

0

100,000 F 2000

0.3

77,000 F 1400

3.0

68,000 F 2500

30

61,000 F 2000

a

14,000 (14)

47,000 F 4900 (61) 31,000 F 1700 (46) 29,000 F 3200 (48)

BM 1.2  105 51,000 F 1400 (51) 61,000 F 500 (79) 50,000 F 3200 (74) 47,000 F 1500 (77)

2.5  105 spleen cells were incubated with 2.5 Ag Con A/ml. a Percent proliferation compared to SpC Con A and the corresponding prednisolone concentration.

was added to the assay containing 2.5  105 BM cells, suppression was reduced to 39%; in this instance the 100% control response is SpC, Con A and 0.3 AM prednisolone. The suppression assay is complicated by the fact that the steroid alone does decrease SpC proliferation [16]. NS cells suppress via the soluble mediator NO [11,13]. We next wanted to determine if prednisolone could also block production of NO by NS cells. Angulo et al. [13] have shown that BM cells produce NO when

incubated in the presence of normal spleen cells and Con A. Fig. 1 shows that cultures containing BM cells, SpC, and Con A produce 38 AM of NO as determined by the Griess assay. The results are plotted as optical density, with the micromoles of NO given as the number at the end of the column. Prednisolone at all three concentrations tested blocked synthesis of NO by BM cells. We next wanted to examine the mechanism by which prednisolone blocks NS cell suppression and NO production. Rodriguez et al. [16] proposed that the steroid acts by blocking the ability of T cells to produce cytokines necessary for NS cell activation. If this is the case, then the addition of exogenous mediators that act directly on the BM cells should restore NO production to cultures that contain prednisolone. BM NS cells can be stimulated to produce NO in the presence of both SpC and Con A [13] or by incubating BM cells with IFN-g and LPS [18]. Fig. 1 shows that addition of LPS and IFN-g to cultures of BM cells, SpC, prednisolone and Con A restored NO production only at the lowest concentration of prednisolone tested. If the prednisolone were only acting on T cells, LPS and IFN-g should restore NO production at all concentrations of prednisolone. Trypan blue exclusion showed no difference in cell viability in cultures that contain prednisolone

Fig. 1. Production of NO by BM cells is inhibited by prednisolone. 1.5  106 BM cells were incubated with 106 SpC and Con A (2.5 Ag/ml). Prednisolone was added at the final concentrations of 30, 3 and 0.3 AM. To some cultures, IFN-g (5 units/ml) and LPS (1 Ag/ml) were also added. Controls contained 1.5  106 normal BM cells without spleen cells. Concentrations of NO (AM) are shown in brackets at the end of the columns. Bars without any NO concentration are comparable to values for TCM alone. Starred (*) groups are compared to N BM, SpC, Con A (**). Values marked with the hatched sign (#), are compared to the control of normal BM plus LPS and IFN-g (##).

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Fig. 2. Prednisolone acts directly on bone marrow cells to inhibit NO production. 1.5  106 BM cells were cultured with LPS (1 Ag/ml) and IFN-g (5 units/ml) for 72 h. Prednisolone was added to some of the wells at the concentrations indicated. Concentrations of NO (AM) are shown at the end of the columns. P values indicate the comparison with the culture of BM cells that contained both IFN-g and LPS.

compared to those that do not (data not shown). Thus, the steroid must have a direct effect on the BM cells, blocking NO synthesis. To establish that prednisolone acts directly on the ability of BM cells to produce NO, BM cells were stimulated with IFN-g and LPS, in the presence or absence of the steroid. Fig. 2 shows that IFN-g and LPS induce BM to produce NO (33 AM); this induction is blocked by prednisolone at all three concentrations tested.

The previous experiments were performed on BM cells from normal mice. Since the ultimate goal is to determine if prednisolone can reverse the effects of LPS in vivo, the next experiment used BM from LPSinjected mice. Fig. 3 shows that BM from LPSinjected mice behaves similarly to normal BM. Both LPS and IFN-g are required to induce NO production from LPS BM cells (49 AM), and again prednisolone is able to block NO production. Comparing Figs. 2 and 3 shows that prednisolone at the lowest concen-

Fig. 3. Prednisolone acts directly on BM from LPS-injected mice to block NO production. LPS was injected into mice; after 48 h, the BM was assayed for its ability to produce NO. 1.5  106 BM cells were cultured with LPS (1 Ag/ml) and IFN-g (5 units/ml). Prednisolone was added to some of the wells, at the indicated concentrations. Concentrations of NO (AM) are shown at the end of the columns. P values indicate the comparison with the culture of BM cells that contained both IFN-g and LPS. The O.D. for TCM alone was 0.058 F 0.005.

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Fig. 4. Prednisolone and LPS act synergistically to induce bone marrow NS cell activity. Mice were injected with nothing, prednisolone (20 Ag/g), LPS (0.5 Ag/gm), or a combination of prednisolone and LPS. Forty-eight hours after the LPS injection, the bone marrow was removed and assayed for the ability to suppress T cell proliferation to the mitogen Con A. The response of 2.5  105 SpC alone was 107,000 CPM.

tration (0.3 Ag) had a greater effect in blocking NO production in normal BM (Fig. 2) compared to BM from LPS-injected mice (Fig. 3). These results are consistent with the fact that BM from LPS-injected mice contains greater numbers of NS cells and produces higher quantities of NO compared to normal BM.

In the next series of experiments, we wanted to determine if prednisolone could reverse the effects of LPS in vivo. Mice received two injections of prednisolone; the first was 1 h before LPS injection, and the second 24 h after LPS injection. Forty-eight hours after receiving LPS, the mice were sacrificed and the BM assayed for its ability to suppress SpC prolifer-

Fig. 5. Bone marrow from mice injected with prednisolone and LPS produce the greatest amount of NO. Mice were injected with nothing, prednisolone (20 Ag/g), LPS (0.5 Ag/gm), or a combination of prednisolone and LPS. Forty-eight hours after the LPS injection, the bone marrow was removed and assayed for the ability to produce NO. 1.5  106 BM cells were cultured with 106 normal spleen cells and the mitogen Con A for 48 and 72 h; the supernatants were then assayed for NO. Concentrations of NO (AM) are shown at the end of the columns.

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Fig. 6. Prednisolone and LPS act synergistically to induce bone marrow NS cell activity. Mice were injected with nothing, prednisolone (0.2 Ag/g), LPS (0.5 Ag/gm), or a combination of prednisolone and LPS. Forty-eight hours after the LPS injection, the bone marrow was removed and assayed for the ability to suppress T cell proliferation to the mitogen Con A. The response of 2.5  105 SpC alone was 120,000 CPM.

ation and produce NO. In the first experiment, mice received a high dose of prednisolone (20 Ag/g). Fig. 4 shows the results from the suppression assay. As previously reported, BM from LPS-injected mice suppresses T cell proliferation to a greater extent than does normal BM; this is most evident at the two highest cell doses. While BM from mice injected with prednisolone alone was slightly less suppressive than normal BM, this is most apparent at the 1.2 and 0.6  105 cell doses. Surprisingly, BM cells from mice that received injections of both LPS and prednisolone

were much more suppressive than even BM from LPS-injected mice. 3  104 BM cells from mice that received both LPS and prednisolone suppressed T cell proliferation by 74% ( p < 0.008 compared to BM from an LPS-injected mouse). Fig. 5 shows that NO production of the BM cells correlates with suppression. BM cells from mice injected with LPS and prednisolone produced more NO than the other cell populations. Fig. 6 shows the results of prednisolone injection into mice at a lower dose (0.2 Ag/g). Again, injecting

Fig. 7. Bone marrow from mice injected with prednisolone and LPS produce the greatest amount of NO. Mice were injected with nothing, prednisolone (0.2 Ag/g), LPS (0.5 Ag/gm), or a combination of prednisolone and LPS. Forty-eight hours after the LPS injection, the bone marrow was removed and assayed for the ability to produce NO. 1.5  106 BM cells were cultured with 106 normal spleens cells and the mitogen Con A for 48 and 72 h; the supernatants were then assayed for NO. Concentrations of NO (AM) are shown at the end of the columns.

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LPS alone increased suppression compared to normal BM. However, in contrast to the high dose, the 0.2 Ag/ g dose did not decrease BM suppression when injected alone. The low dose of prednisolone acted in a synergistic manner with LPS to increase the ability of the BM cells to suppress proliferation; this is most evident at the lower doses of BM cells. Fig. 7 shows that the ability of BM cells to suppress proliferation correlates with NO production. BM from mice injected with both LPS and prednisolone produce the most NO. It should be noted that the spleen of a mouse receiving an LPS injection is enlarged; prednisolone at the high dose (20 Ag/g) reversed this effect. The high dose of prednisolone also resulted in thymic involution. Neither observation was seen when the low dose of prednisolone was used.

4. Discussion Injecting LPS into mice increases BM NS cell activity, as measured by the ability to suppress SpC proliferation and production of NO in vitro [7,13]. The focus of this study was to determine if the glucocorticoid prednisolone could reverse the effect of LPS in vivo. Previously, Rodriguez et al. [16] have shown that steroids could inhibit the ability of NS cells to suppress T cell proliferation in vitro; this effect was reversed by the addition of IFN-g to the cultures. The authors concluded that the steroid acted by blocking cytokine production. We hypothesized that injecting the immunosuppressive agent prednisolone, along with LPS, would block the synthesis of pro-inflammatory cytokines necessary for activating NS cells in vivo. Rodriguez et al. [16] were unable to examine the direct effect of glucocorticoids on BM NS cells. Subsequent studies have shown that NO is the primary suppressive molecule produced by NS cells, and NO production can be stimulated in the presence of soluble mediators [11,13,18] making it possible to study the effect of prednisolone on NS cells directly. Figs. 2 and 3 demonstrate that prednisolone can block synthesis of NO by NS cells. Using NO production as the measure for NS activity is more selective, compared to suppression, since BM cells can be stimulated to produce NO in the absence of SpC. Prednisolone inhibits NO

production by BM cells from both normal and LPSinjected mice. This is the first report that steroids can block production of NO by BM NS cells. This finding is consistent with reports showing that steroids can block both cytokine production by T cells and NO production by macrophages. Glucocorticoids have been shown to prevent translocation of the transcriptional factor NF-nB from the cytoplasm to the nucleus, thus blocking transcription of the NOS2 enzyme [19]. We next wanted to determine if prednisolone could block the activity of NS cells in vivo. Figs. 4 –7 show that BM cells from mice receiving injections of both LPS and prednisolone were the most suppressive and produced the most NO even compared to mice receiving LPS alone. The result that mice receiving injections of both steroid and LPS developed greater NS activity was unexpected. There are numerous reports of corticosteroids used to mitigate the effect of endotoxemia in vivo [14,21]. Studies have shown that glucocorticoids block the ability of macrophages, endothelial cells and hepatocytes to produce NO in responses to LPS in vivo [19 –21]. Most studies look at a narrow time frame after the injection of LPS and steroid, usually within minutes up to 20 h [6]. In these studies for the steroid to be effective, it must be administered approximately 1 h before LPS [22]. Because of the 48-h time frame used in the current study, we injected mice twice with steroid, once before and again 18 h after receiving LPS. BM was then removed and assayed either for the ability to produce NO or to suppress T cell proliferation. The ability of glucocorticoids to enhance NO production is not unique to NS cells. Yamada et al. [23] have shown that proliferation of rat thymocytes was reduced in animals receiving hydrocortisone 48 h prior to removal of the thymus. Proliferation could be restored by the addition of the NOS inhibitor LMMA to the cultures. The mechanism by which prednisolone and LPS act to enhance NS cell activity, in vivo, is not known. One possibility is that the steroid inhibits the synthesis of a cytokine(s) that normally down-regulates NS cell activity [24]. This seems unlikely, since prednisolone, at the highest dose, actually decreased NS activity when injected alone. A second possibility is that the steroid acts by up-regulating cytokine receptors on the surface of the NS cells [22,24]. It is possible that prednisolone is stimulating synthesis of cytokine receptors on the surface of the NS cells, while the

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LPS induces production of the appropriate cytokine to bind the receptor. This theory is inconsistent with the in vitro data, showing that prednisolone acts directly on the NS cells to block NO production. A third possibility is that prednisolone and LPS may each induce synthesis of a separate cytokine that act together in vivo to stimulate NS cells. Steroids have been shown to induce synthesis of some cytokines [22,24]. Again this hypothesis is not totally consistent with the in vitro data showing prednisolone to have a suppressive effect on NO generation. The mechanism for enhanced NS cell activation after injection of LPS and prednisolone remains to be determined. Bacterial sepsis is the 13th leading cause of death in the United States. The incidence of sepsis is estimated to be 300,000 cases per year, with a 20 –30% mortality rate [1]. Sepsis, especially caused by Gram-negative bacteria, can lead to manifestations such as fever, shock, disseminated intravascular coagulation and multiple organ failure [3]. Clinically, corticosteroids are used as one treatment for endotoxemia. Presumably, the steroids act by blocking soluble mediators that are produced during sepsis [20,21]. However, some reports suggest that the administration of glucocorticoids is contraindicated for treatment of sepsis [1,22]. While our results show a synergistic effect between LPS and prednisolone on BM NS activity, it is unclear what effect this may have on the outcome of endotoxemia. Reports show that elevated levels of NO are associated with endotoxemia and treatment with inhibitors of NO synthesis enhance survival [5,26]. In contrast, knockout mice for NOS2 did not show any difference in susceptibility to LPS injections, compared to normal controls [25]. In addition to endotoxemia, steroids are used to treat a wide array of inflammatory responses such as asthma, rheumatoid arthritis, and graft vs. host disease. The results presented here illustrate the complex interactions that take place between steroids and cells of the immune system. They also indicate the difficulty in predicting the outcome of steroid treatment based on in vitro results.

Acknowledgements This work was supported by the University of Akron Faculty Research Grant 1464.

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