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The Journal of Immunology, 2004, 172: 4834-4843.
Copyright © 2004 by The American Association of Immunologists

Cytokine Dysregulation Induced by Apoptotic Cells Is a Shared Characteristic of Macrophages from Nonobese Diabetic and Systemic Lupus Erythematosus-Prone Mice 1

Hanli Fan, Angelika Longacre, Fanyong Meng, Vimal Patel, Kevin Hsiao, Jason S. Koh and Jerrold S. Levine2

Section of Nephrology, Department of Medicine, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1{beta} and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1{beta} by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The etiology of autoimmunity remains elusive. One reason for this is that most of the major autoimmune diseases, including type I diabetes mellitus (DM)3 and systemic lupus erythematosus (SLE), have polygenic origins (1, 2, 3). Linkage analysis and genetic mapping studies in murine models of these two diseases suggest that as many as 20 genes contribute to the development of type I DM in mice (2), while as many as 30 genes contribute to the development of SLE (3). Elucidating the genetic origins of autoimmunity is further complicated by the fact that diseases such as SLE almost certainly represent syndromes, with multiple different underlying genetic abnormalities or environmental factors leading to a common final phenotype. In understanding the role of individual genes, it is important to recognize that different loci may contribute to different clinical features, laboratory abnormalities, or even stages of disease (3, 4). For example, it seems reasonable to assume that certain genes may provide a permissive background upon which autoimmunity is more likely to occur, whereas other genes may determine whether the resultant autoimmunity is systemic, as in SLE, or restricted to one organ, as in DM.

Macrophages (M{phi}) from nonobese diabetic (NOD) mice, which spontaneously develop autoimmune DM, share a specific defect in elicited cytokine production (5, 6, 7, 8) with M{phi} from multiple diverse strains of SLE-prone mice (9, 10, 11, 12, 13). Peritoneal and bone marrow-derived M{phi} from all these strains underexpress the cytokine IL-1 in response to LPS stimulation (5, 6, 7, 8, 9, 10, 11, 12, 13). In the case of SLE-prone MRL M{phi}, we have demonstrated the following features of this abnormality. As shown by the construction of irradiation chimeras in which bone marrow from SLE-prone mice was transferred into lethally irradiated normal recipients, and vice versa, IL-1 underexpression is intrinsic to the M{phi} and independent of the host autoimmune environment (10). Moreover, the defect is fully manifest in M{phi} from mice as young as 1 wk of age (10), and neither the pattern nor the magnitude of the defect changes with age or the development of disease (10, 13). Underexpression of IL-1 exists at both the mRNA and protein level. It is transcriptionally determined, and equally affects secreted and cell-associated IL-1 bioactivity (9, 10, 11, 12, 13). The defect is progressive over time, increasing in magnitude from ~2-fold within the first 24 h of culture to as much as 50-fold by 24–48 h (9, 10, 11, 12, 13). Finally, the defect is not limited to induction of IL-1 by LPS, but also occurs in response to stimulation by other danger signals and M{phi} activators, including toxic shock syndrome toxin-1, lipoteichoic acid, and TNF-{alpha} (13).

We have recently shown that SLE-prone M{phi} are able to respond normally to LPS, but that an unidentified lipid factor(s) in FBS results in inhibition of LPS-induced IL-1 expression (13). Thus, when SLE-prone M{phi} are cultured in medium containing either no FBS or FBS that has been delipidated (dFBS), expression of IL-1 is completely normal (13). Significantly, exposure to apoptotic cells or anionic lipids, such as those expressed on the surface of apoptotic cells, elicits the identical abnormality (13). Affected SLE-prone strains are genetically diverse and include MRL/+, MRL/lpr, New Zealand Black (NZB), New Zealand White (NZW), NZB/W F1, BXSB, and LG/J, all of which express or contribute to the development of autoimmunity (13). In contrast, no similar defect can be found in 13 nonautoimmune control strains (9, 10, 11, 12, 13). Elicitation of this defect by exposing SLE-prone M{phi} to apoptotic cells or serum lipids led to the dysregulated expression of multiple cytokines (13).

The presence of similar defects in cytokine expression in M{phi} from NOD and SLE-prone mice suggests that the genetic basis for this abnormality may represent a shared background predisposing to autoimmunity. To address this hypothesis, we determined whether dysregulated cytokine expression by NOD M{phi} is also dependent on FBS lipids, and whether this defect can be elicited by exposure of NOD M{phi} to apoptotic cells. We report in this work that M{phi} from NOD mice indeed respond abnormally to serum lipids and apoptotic cells, mimicking closely in nearly all aspects the apoptotic cell-dependent abnormality we have observed in M{phi} from multiple SLE-prone strains. Given the increasing role that apoptotic cells and Ags have been shown to play in immune homeostasis, a signaling abnormality that is triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6, DBA/2J (DBA.2), KK/HlJ (KK), and NOD/LtJ (NOD) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/KsOlaHsd-leprdb (db/db) and C57BL/6OLAHSD-lepob (ob/ob) were purchased from Harlan Sprague-Dawley (Madison, WI). Unless otherwise indicated, these mice were male and used between 4 and 6 wk of age. Diseased NOD mice, generously provided by Y.-Y. Fu (University of Chicago, Chicago, IL), were LIGHT (TNF superfamily 14) transgenic NOD retired male breeders, greater than 6 mo of age, with blood glucose levels above 250 mg/100 ml (14 mM) (14). Mice were maintained in a specific pathogen-free facility. All animal protocols were approved by the Institutional Animal Care and Use Committee at University of Chicago.

M{phi} culture

Peritoneal exudate cells were harvested by lavage 3 days after i.p. injection of 1.5 ml of 4.05% thioglycolate broth (9, 10, 11, 12, 13). Cells were washed twice in RPMI 1640 and plated in 60 x 15-mm tissue culture dishes at 4 x 106 cells/dish in R.0 culture medium (RPMI 1640 with 2 mM L-glutamine, 5 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin). After incubation at 37°C for 4 h, nonadherent cells were removed by washing with RPMI 1640. The remaining adherent cells, >98% M{phi} as determined by morphologic examination and nonspecific esterase staining, were cultured in either FBS-free R.0 or R.10 (R.0 plus 10% FBS) medium (9, 10, 11, 12, 13). M{phi} were used after overnight culture in their respective medium.

FBS delipidation

FBS was delipidated by one of two protocols. FBS was mixed 1:1 (v/v) for 2 h at 22°C with either butanol/diethyl ether (1:4, v/v) (15) or chloroform/methanol (2:1, v/v) (16). The organic and aqueous phases were separated by allowing the mixture to sit overnight at 22°C (butanol/diethyl ether) or by high-speed centrifugation (30,000 x g) for 30 min at 22°C. Residual organic solvents were removed from the delipidated aqueous phase by blowing a stream of nitrogen gas across the liquid surface. The resultant dFBS was sterilely filtered for later use.

Thymocyte harvest and induction of apoptosis

Apoptosis was induced in freshly isolated murine thymocytes by incubation for 6 h with 5 x 10−6 M hydrocortisone in either R.10 or R.0 medium (13, 17, 18). Before addition to M{phi}, thymocytes were washed three times in RPMI 1640 and resuspended in R.0 medium. Viable thymocytes were defined as propidium iodide (PI)-negative cells with faint nuclear Hoechst staining. Apoptotic thymocytes were defined as PI-negative cells with bright nuclear Hoechst staining and decreased cell size. Postapoptotic thymocytes (i.e., apoptotic thymocytes that had lost cell membrane integrity) were defined as PI-positive cells with bright Hoechst staining and decreased cell size. By these criteria, >=60% of thymocytes were apoptotic, ~15% were viable, and ~25% were postapoptotic. The kinetics of induction of apoptosis and the population distribution at 6 h were identical for thymocytes incubated in R.10 vs R.0 medium. Necrotic cells, as defined by increased cell size in association with uptake of PI and faint Hoechst staining, comprised <0.1% of the final cell population.

RNA isolation

Total RNA was isolated from M{phi} by phenol-chloroform extraction after lysis of cells in 1:1 mixture of water-saturated phenol and solution D (4 M guanidine isothiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 102 µM 2-ME) containing 120 mM sodium acetate, pH 4.0. All procedures were performed on ice or at 4°C.

RNase protection assays

RNase protection assays were performed using a RiboQuant In Vitro Transcription Kit (BD PharMingen, San Diego, CA), according to manufacturer’s instructions. All probes were for murine cytokines.

ELISA for secreted IL-1{beta}

M{phi} culture and stimulation with LPS (100 ng/ml) were identical with that for RNase protection assays. At 0, 8, 24, and 48 h after stimulation, the conditioned medium was removed and replaced with fresh medium and LPS. Conditioned medium collected at 24 and 48 h was assayed for secreted IL-1{beta} by ELISA using a mouse IL-1{beta} Enzyme Immunometric Assay Kit (Assay Designs, Ann Arbor, MI), according to manufacturer’s instructions.

Immunoblotting for total cellular IL-1{beta}

M{phi} culture and stimulation with LPS (100 ng/ml) were identical with that for RNase protection assays. Whole cell lysates (20 µg per sample) were separated by 10% SDS-PAGE under reducing conditions, transferred onto polyvinylidene difluoride membranes, and probed for IL-1{beta} using polyclonal goat anti-mouse IL-1{beta} IgG, according to manufacturer’s specifications (R&D Systems, Minneapolis, MN).

Protein assay of cytokine expression

M{phi} culture and stimulation with LPS (100 ng/ml) were identical with that for RNase protection assays. Protein assays were performed using the RayBio Mouse Cytokine Array Kit (RayBiotech, Norcross, GA), according to manufacturer’s instructions. In brief, after stimulation with LPS (100 ng/ml) for 24 h, M{phi} cell lysates were assayed on a membrane support containing Abs specific for a panel of murine cytokines and chemokines. Ab-captured cytokines and chemokines were detected by biotinylated Abs, followed by labeled streptavidin. Separate membranes were used for each experimental condition. Positive controls on each membrane were used to normalize all results and enable comparison among different membranes. All cytokine and chemokine levels are expressed as a percentage of the intensity of the positive control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Underexpression of IL-1 by M{phi} from autoimmune DM-prone NOD mice is dependent upon the presence of FBS

As previously reported (5, 6, 7, 8, 9, 10, 11, 12, 13), peritoneal M{phi} from prediseased autoimmune DM-prone NOD mice demonstrated a profound defect in LPS-induced expression of IL-1{beta} (Fig. 1, A and B). The difference in IL-1{beta} expression between nonautoimmune DBA.2 and NOD M{phi} increased with time. Expression by NOD M{phi} at 8 h was only slightly less than that by DBA.2 M{phi} (Fig. 1A). Although DBA.2 M{phi} maintained expression of IL-1{beta} through 24 and 48 h (72 and 25% of that at 8 h), expression of IL-1{beta} by NOD M{phi} was virtually undetectable at 24 and 48 h (4.0 and ~0% of that at 8 h) (Fig. 1, A and B). Essentially the same results were obtained when we used C57BL/6 mice as a control strain (data not shown).



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  		FIGURE 1. IL-1{beta} underexpression by M{phi} from NOD mice is dependent on the presence of FBS. A–C, Primary cultures of thioglycolate-elicited peritoneal M{phi} from DBA.2 and NOD mice were cultured in the presence (FBS) or absence (FBS-free) of 10% FBS. After overnight culture, M{phi} were stimulated continuously with LPS (100 ng/ml) for the indicated times (0, 8, 24, or 48 h). Total cellular RNA was harvested, and RNase protection assays (BD PharMingen) were performed for murine IL-1{beta} and the housekeeping genes GAPDH and ribosomal L32. A, Shown are representative blots for murine IL-1{beta} from seven independent experiments. B and C, Relative expression of IL-1{beta} message in the presence (B) or absence (C) of FBS was determined by densitometric scanning and normalized for L32. The density at 8 h for DBA.2 and NOD M{phi} was arbitrarily set at 100%. Error bars denote mean ± SE.

 
In multiple experiments, NOD M{phi} only rarely revealed detectable IL-1{beta} message at 48 h, even upon prolonged exposure of blots from RNase protection assays. We have chosen blots with relatively long exposure times that emphasize the striking lack of IL-1{beta} message at the 24- and 48-h time points (Fig. 1A). A similar principle guided our choice of blots in all later figures.

Strikingly, removal of FBS from the culture medium (FBS free) resulted in equalization of IL-1{beta} mRNA expression between NOD and control DBA.2 M{phi} (Fig. 1, A and C). In the absence of FBS, expression of IL-1{beta} by NOD M{phi} was virtually identical with that of DBA.2 M{phi} at all time points (Fig. 1, A and C). The inhibitory effect of FBS on IL-1{beta} expression by NOD M{phi} was dose dependent (Fig. 2). Inhibition was observed with as low a concentration of FBS as 2%, and was fully manifest at a concentration of 4%.



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  		FIGURE 2. The inhibitory effect of FBS on IL-1{beta} underexpression by NOD M{phi} is dose dependent. Peritoneal M{phi} from the indicated strains were cultured in the presence of varying concentrations of FBS. Total cellular RNA was harvested 24 h after continuous stimulation with LPS (100 ng/ml), and RNase protection assays (BD PharMingen) were performed for murine IL-1{beta} and the housekeeping genes GAPDH and ribosomal L32. Shown are representative blots for murine IL-1{beta} from three independent experiments.

 
FBS-dependent inhibition of IL-1{beta} expression by NOD M{phi} was also evident at the level of protein expression, as evidenced by both immunoblotting for total cellular IL-1{beta} (Fig. 3A) and ELISA for secreted IL-1{beta} (Fig. 3B). Just as for IL-1{beta} message, removal of FBS from the culture medium led to equalization of IL-1{beta} protein between NOD and control M{phi} (Fig. 3).



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  		FIGURE 3. FBS-dependent underexpression of IL-1{beta} by NOD M{phi} occurs at a protein level. Peritoneal M{phi} from DBA.2, C57BL/6, and NOD mice were cultured in the presence (FBS) or absence (FBS-free) of 10% FBS. After overnight culture, M{phi} were stimulated continuously with LPS (100 ng/ml) for the indicated times (0, 8, 24, or 48 h). A, Whole cell lysates obtained at the indicated times were immunoblotted for total cellular murine IL-1{beta}. Ponceau S staining confirmed equal loading of gels and equal transfer to polyvinylidene difluoride membranes. By densitometry, the relative expression of IL-1{beta} protein by NOD M{phi} in the presence of FBS was 0.65, 0.17, and <0.05 that of control M{phi} at 8, 24, and 48 h. B, Conditioned medium (CM) collected at the indicated times was assayed for secreted IL-1{beta} by ELISA (Assay Designs). CM collected at 24 h represents IL-1{beta} secreted from 8 to 24 h after LPS stimulation, while CM collected at 48 represents IL-1{beta} secreted from 24 to 48 h after LPS stimulation. Error bars denote mean ± SE from three experiments.

 
Together, these data indicate that NOD M{phi} are able to respond normally to LPS, but that an unidentified factor(s) in FBS results in inhibition of LPS-induced IL-1{beta} expression. It should be noted that FBS-free culture also increased the magnitude of IL-1{beta} expression by nonautoimmune DBA.2 M{phi}, although the degree of change was far less than that seen for NOD M{phi}. Thus, it is possible that the response of NOD M{phi} to FBS may represent an exaggeration of a normal regulatory response.

Murine strains prone to type I (autoimmune), but not type II (nonautoimmune), DM demonstrate FBS-dependent IL-1{beta} underexpression

Female NOD mice develop DM at an earlier age and at a higher frequency than do male NOD mice (4). Despite these differences, FBS-dependent IL-1 underexpression occurred to an equivalent degree in M{phi} from prediseased male and female NOD mice (Fig. 4A). Importantly, the defect was related to autoimmunity rather than to DM, as none of the three strains susceptible to type II (nonautoimmune) DM (KK, db/db, and ob/ob) (19, 20, 21, 22) showed a similar defect (Fig. 4B). Finally, neither of the two nonautoimmune strains tested in this study (C57BL/6 and DBA.2) (Fig. 4C) nor 11 nonautoimmune strains evaluated previously (A/J, AKR/J, A.Thy, B.10, B.10A, B.10BR, BALB/c, C3HeB/FeJ, C3H/HeN, CBA/J, and SWR) (13) showed a similar defect. As opposed to M{phi} from nonautoimmune strains, NOD M{phi} are characterized by the virtual absence of detectable message for IL-1{beta} at 24 and 48 h, even after prolonged exposure of blots.



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  		FIGURE 4. Murine strains prone to type I (autoimmune), but not type II (nonautoimmune), DM demonstrate FBS-dependent IL-1{beta} underexpression. A–C, Peritoneal M{phi} from the indicated strains were cultured in the presence or absence of 10% FBS. Total cellular RNA was harvested at the indicated times after continuous stimulation with LPS (100 ng/ml), and RNase protection assays (BD PharMingen) were performed for murine IL-1{beta} and the housekeeping genes GAPDH and ribosomal L32. Shown are representative blots for murine IL-1{beta} from at least three independent experiments. D and E, Relative expression of IL-1{beta} message at 8 and 48 h was determined by densitometric scanning for each strain and normalized for ribosomal L32. The ratio of expression at 48 h to that at 8 h was calculated and plotted for both FBS-containing (D) and FBS-free (E) conditions. Also included for comparison are previously published data obtained from 8 control and 10 SLE-prone strains (13 ). *, Indicates new data obtained as part of this study.

 
This property of NOD M{phi} is represented graphically in Fig. 4, D and E, in which the relative expression of IL-1{beta} at 48 h, normalized to that at 8 h, is shown for all strains under both FBS-containing (Fig. 4D) and FBS-free (Fig. 4E) conditions. We also included data from our previous study (13) on SLE-prone strains to highlight the similarity of the defects seen in M{phi} from NOD and SLE-prone mice. Under FBS-containing conditions, relative expression at 48 h was 25.6 ± 4.1% for the five nonautoimmune strains examined in this study (C57BL/6, DBA.2, KK, db/db, ob/ob) and ~0% for NOD mice (p < 0.005). In marked contrast, when M{phi} were cultured in the absence of FBS, there was no statistical difference in expression at 48 h between nonautoimmune and NOD strains (87.1 ± 6.3% vs 87.7 ± 4.6%, p > 0.90). Under FBS-containing conditions, relative expression of IL-1{beta} at 48 h was 38.5 ± 7.8% for all nonautoimmune strains and 1.3 ± 0.5% for autoimmune DM- and SLE-prone strains (p < 0.0001). In contrast, when M{phi} were cultured in the absence of FBS, there was no difference in expression of IL-1{beta} at 48 h between autoimmune and nonautoimmune strains (85.6 ± 5.7% vs 81.7 ± 5.4%, p > 0.60). These data again highlight the similarity of the defects found in M{phi} from NOD and SLE-prone mice.

It should be stressed that, except where indicated, only young prediseased NOD mice were used as a source of M{phi}. This was done to minimize any effects of disease, thereby enabling us to distinguish those abnormalities that may play a fundamental role in the etiology of autoimmune DM from those that are a consequence of established disease. Our results therefore suggest that FBS-dependent IL-1{beta} underexpression is independent of disease progression and may represent a background susceptibility trait in NOD mice. Several additional features of this defect bear on this point. First, while male and female NOD mice differ in the age of onset and prevalence of DM (4), the pattern and magnitude of the defect was essentially the same, irrespective of sex (Fig. 4, A and D). This implies that the basis for this defect is independent of sex-related genes that modulate the course of autoimmune DM. Second, the presence and magnitude of the defect were unaffected by disease and its sequelae. Thus, FBS-dependent IL-1{beta} underexpression was equally manifest in M{phi} from both diseased and prediseased NOD mice (Fig. 4, A and D). Finally, the features of this defect segregate with autoimmunity, being equally manifest in M{phi} from NOD and SLE-prone strains, but absent in M{phi} from 3 nonautoimmunne DM-prone strains and 13 control strains. Taken together, these results suggest that FBS-dependent IL-1{beta} underexpression represents a shared phenotype of murine autoimmunity.

FBS lipids are necessary to elicit IL-1{beta} underexpression by NOD M{phi}

We next sought to identify the specific FBS-dependent factor(s) that elicits this defect in NOD M{phi}. Delipidation of FBS, by two distinct methods (15, 16), restored IL-1{beta} expression by NOD M{phi} to nearly normal levels at 24 and 48 h, while NOD M{phi} exposed to untreated FBS showed reduced levels of IL-1{beta} (Fig. 5). As this lipid-dependent effect was observed with NOD, but not DBA.2 M{phi}, these data demonstrate that a lipid or lipids found in FBS is responsible for IL-1{beta} underexpression by NOD M{phi}.



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  		FIGURE 5. FBS lipids are necessary to elicit IL-1{beta} underexpression by NOD M{phi}. Peritoneal M{phi} from DBA.2 and NOD mice were cultured in the presence of 10% FBS, either untreated or delipidated by one of two methods (dFBS1 or dFBS2) (15 16 ). Total cellular RNA was harvested at the indicated times after continuous stimulation with LPS (100 ng/ml), and RNase protection assays (BD PharMingen) were performed for murine IL-1{beta} and the housekeeping genes GAPDH and ribosomal L32. Shown are representative blots from three independent experiments.

 
Apoptotic cells elicit underexpression of IL-1{beta} by NOD M{phi}

We have previously shown that opsonized apoptotic cells can substitute for FBS in reproducing the defect in IL-1{beta} expression in SLE-prone M{phi} (13). We therefore determined the effect of apoptotic cells on IL-1{beta} expression by NOD M{phi}.

Unopsonized apoptotic thymocytes alone (i.e., thymocytes induced to undergo apoptosis in FBS-free medium) were an incomplete stimulus and failed to elicit a defect by NOD M{phi} (Fig. 6). However, when thymocytes were induced to undergo apoptosis in the presence of FBS and then extensively washed, they yielded a significant defect by NOD M{phi}. Importantly, while neither dFBS nor nonopsonized apoptotic thymocytes alone could reproduce the defect, apoptotic thymocytes opsonized with dFBS yielded a powerful defect. This result indicates that interaction of a nonlipid FBS factor, presumably a protein, with the surface of apoptotic cells creates a ligand capable of eliciting the defect. As a final control, viable thymocytes (alone or washed after preincubation with FBS) were unable to produce a defect. Because viable thymocytes eventually undergo apoptosis during prolonged culture, this last result suggests that the necessary nonlipid factor in FBS interacts specifically with the surface of apoptotic, but not viable, thymocytes. IL-1{beta} expression by M{phi} from six other representative nonautoimmune strains (AKR/J, BALB/c, C3H/HeN, C57BL/6, CBA/J, and SWR) resembled that of DBA.2 M{phi} and was not down-regulated in response to dFBS-opsonized apoptotic thymocytes (data not shown) (13). Finally, the effect of dFBS-opsonized apoptotic cells on IL-1{beta} expression by NOD M{phi} was dose dependent over a range of apoptotic cell to M{phi} ratios extending from 1:1 to 10:1 (Fig. 7).



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  		FIGURE 6. Apoptotic cells elicit underexpression of IL-1{beta} by NOD M{phi}. Peritoneal M{phi} (4 x 106) from DBA.2 and NOD mice were cultured in FBS-free medium containing 10% FBS, 10% dFBS1, or 40 x 106 thymocytes. Apoptotic thymocytes were produced by a 6-h incubation with 5 x 10−6 M hydrocortisone in FBS-free medium alone ({Phi}) or containing 10% FBS (FBS), 10% dFBS1 (dFBS1), or 10% dFBS2 (dFBS2). Viable thymocytes (FBS) were similarly handled, but in the absence of hydrocortisone. Before addition to M{phi}, all thymocytes (including those that had been induced to undergo apoptosis in the presence of FBS or dFBS) were extensively washed with FBS-free medium. Total cellular RNA was harvested at the indicated times after continuous stimulation with LPS (100 ng/ml), and RNase protection assays (BD PharMingen) were performed for murine IL-1{beta} and the housekeeping genes GAPDH and ribosomal L32. Shown are representative blots for murine IL-1{beta} from three independent experiments.

 


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  		FIGURE 7. The inhibitory effect of apoptotic cells on IL-1{beta} underexpression by NOD M{phi} is dose dependent. Peritoneal M{phi} (4 x 106) from the indicated strains were cultured in FBS-free medium containing the indicated number of apoptotic thymocytes. Apoptosis was induced by a 6-h incubation with 5 x 10−6 M hydrocortisone in FBS-free medium containing 10% dFBS. Before addition to M{phi}, thymocytes were extensively washed with FBS-free medium. After overnight culture, M{phi} were stimulated with LPS (100 ng/ml) for 24 h. RNase protection assays (BD PharMingen) were performed for murine IL-1{beta} and the housekeeping genes GAPDH and ribosomal L32. Shown are representative blots for murine IL-1{beta} from three independent experiments.

 
FBS-dependent abnormalities in NOD M{phi} extend to multiple other cytokines in addition to IL-1{beta}

We next determined whether M{phi}-derived cytokines other than IL-1{beta} show a similar FBS-dependent defect in LPS-induced expression (Fig. 8). As compared with DBA.2 and C57BL/6 M{phi}, cytokine expression by NOD M{phi} in the presence of FBS was decreased in duration and magnitude for some cytokines (IL-1{beta}, IL-1 receptor antagonist, IL-6, IL-12 p35, GM-CSF, M{phi} inflammatory protein-1{beta} (MIP-1{beta}), TNF-{alpha}), but not other cytokines (IL-12 p40, M-CSF, M{phi} migration inhibition factor, RANTES, TGF-{beta}1, TGF-{beta}2, TGF-{beta}3). In all cases, FBS-free culture led to equalization of LPS-induced expression between NOD and nonautoimmune DBA.2 or C57BL/6 M{phi}.



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  		FIGURE 8. FBS-dependent abnormalities extend to multiple other cytokines in addition to IL-1{beta}. A and B, Peritoneal M{phi} from DBA.2, C57BL/6, and NOD mice were cultured in the presence or absence of 10% FBS. Total cellular RNA was harvested at the indicated times after continuous stimulation with LPS (100 ng/ml), and RNase protection assays (BD PharMingen) were performed for the indicated murine cytokines. Breaks in the blot represent different exposure times for optimal presentation of data. No IL-4 and IFN-{gamma} were detected, even after prolonged exposure, confirming the purity of M{phi} cultures. Shown are representative blots from four independent experiments.

 
We confirmed the results of RNase protection by protein assay for four of these cytokines. In the presence of FBS, protein expression of IL-6, IL-12 p70, and TNF-{alpha} was decreased in NOD M{phi} as compared with DBA.2 and C57BL/6 M{phi} (Fig. 9A), whereas, in the absence of FBS, protein expression of these three cytokines was equalized (Fig. 9A). The fourth cytokine, RANTES, showed no defect in expression at either an mRNA or protein level of expression (Fig. 9A). Combining the results of Figs. 3 and 9, we have therefore confirmed by protein assay the existence of a FBS-dependent defect in the expression of four cytokines (IL-1{beta}, IL-6, IL-12, and TNF-{alpha}).



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  		FIGURE 9. FBS-dependent abnormalities in cytokine and chemokine expression by NOD M{phi} occur at a protein level. A and B, Peritoneal M{phi} from NOD and control (DBA.2, C57BL/6) mice were cultured in the presence or absence of 10% FBS. Cellular lysates were prepared after 24 h of continuous stimulation with LPS (100 ng/ml). Protein expression was determined by immunoassay on a membrane support containing Abs specific for a panel of murine cytokines and chemokines (RayBiotech). A, Relative expression for each cytokine or chemokine was determined by densitometric scanning and normalized as percentage of signal intensity of a positive control included on each membrane. Error bars denote mean ± SE of two experiments. B, The ratio of protein expression by NOD vs control M{phi} was calculated for each of the seven cytokines showing FBS-dependent underexpression, and plotted for both FBS-containing (R.10) and FBS-free (R.0) conditions.

 
Finally, by protein assay, we showed a FBS-dependent defect in the expression of several chemokines not tested by RNase protection assay. These included cutaneous T cell attractant chemokine (CTACK), monocyte chemoattractant protein-1 (MCP-1), MIP-2, and secondary lymphoid tissue chemokine (SLC, also known as 6Ckine) (Fig. 9A). These protein data are summarized in Fig. 9B. As compared with DBA.2 and C57BL/6 M{phi}, the relative expression of IL-6, IL-12, TNF-{alpha}, CTACK, MCP-1, MIP-2, and SLC by NOD M{phi} in the presence of FBS was 0.15 ± 0.05. In contrast, when M{phi} were cultured in the absence of FBS, the relative expression of these same cytokines by NOD M{phi} was fully corrected to 1.36 ± 0.14 (p < 0.005 by paired t test, FBS vs FBS free).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that peritoneal M{phi} from prediseased NOD mice, a model for type I (autoimmune) DM, demonstrate a defect in cytokine expression strikingly similar to that of M{phi} from prediseased mice of the major murine models of SLE (MRL/+, MRL/lpr, NZW, NZB, NZW/B F1, BXSB, and LG/J) (4, 5, 6, 7, 8, 9, 10, 11, 12, 13). This defect, which is characterized by IL-1{beta} underexpression in NOD (5, 6, 7, 8) and SLE-prone strains (9, 10, 11, 12, 13), is triggered by FBS or apoptotic cells, and is fully corrected by culture in medium containing either no FBS or dFBS. Although the identity of the lipid components in FBS remains undetermined, the most likely candidates are oxidatively modified lipids, lipoproteins, or lipid-protein adducts, whose uptake by M{phi} relies upon many of the same receptors as do apoptotic cells (23, 24, 25). We did not directly test the role of specific lipids in inducing the defect in NOD macrophages. However, in our earlier studies using M{phi} from SLE-prone strains, we found that neutral phospholipids, such as phosphatidylethanolamine and phosphatidylcholine, did not reproduce the defect (13). In contrast, the addition to FBS-free medium of several anionic lipids, including phosphatidylserine, which is uniquely expressed on the surface of apoptotic cells, resulted in IL-1 underexpression (13).

Elicitation of this defect led to the dysregulated expression of multiple cytokines. Of 18 cytokines examined, the expression of 11 was down-regulated (IL-1{beta}, IL-1 receptor antagonist, IL-6, IL-12 p35, CTACK, GM-CSF, MCP-1, MIP-1{beta}, MIP-2, SLC, TNF-{alpha}), while the expression of the other 7 was unaffected (IL-12 p40, M-CSF, migration inhibition factor, RANTES, TGF-{beta}1, TGF-{beta}2, TGF-{beta}3). In all cases, culture under FBS-free conditions led to equivalent expression of cytokines.

The pattern of cytokine dysregulation in the NOD M{phi} is very similar to that observed for SLE-prone M{phi} (13), with several minor differences. M{phi} from SLE prone, but not NOD, also demonstrated a FBS-dependent defect in the expression of IL-10, IL-12 p40, and RANTES. Although individual RNase protection assays from NOD mice showed FBS-dependent abnormalities in these three cytokines, the results were not consistent. As IL-10 and IL-12 p40 are both expressed in relatively low abundance, this difference between NOD and SLE-prone M{phi} may be more apparent than real, and may reflect a problem with sensitivity.

Importantly, no similar defect in cytokine expression can be found in 16 nonautoimmune strains (5 assessed in the present study, and 11 in our previous study (13)), including 3 strains that develop type II (nonautoimmune) DM. The existence of an identical defect in M{phi} from prediseased mice that develop two autoimmune diseases as distinct as DM and SLE lends strong support to the hypothesis that the genetic basis for this abnormality may constitute a permissive or predisposing background for autoimmunity. We speculate that additional genetic factors determine the targets of autoimmunity, whether systemic, as in SLE, or organ specific, as in DM.

The finding that this defect in cytokine expression is elicited by apoptotic cells in both DM and SLE is extremely important. Considerable evidence suggests a role for apoptotic cells in immune homeostasis. This role is most easily seen via the intimate connection between autoimmunity and apoptosis. Multiple studies, including work from our laboratory, have shown that apoptotic cells and their products are the target of autoantibodies across a broad spectrum of autoimmune diseases, including SLE, scleroderma, antiphospholipid syndrome, and anti-neutrophil cytoplasmic Ab-positive vasculitis (17, 26, 27, 28, 29, 30, 31, 32). Indeed, virtually all of the most prominent self Ags in SLE are known to localize on the surface of apoptotic cells (17, 26, 27, 28, 29, 30, 31, 32). These self Ags include nucleosomal DNA-histone complexes, small nuclear ribonucleoproteins (including the Smith and U1-A Ag), cytoplasmic ribonucleoproteins (SS-A/Ro, SS-A/La), and the target Ags of antiphospholipid autoantibodies (17, 26, 27, 28, 29, 30, 31, 32). Moreover, immunization of nonautoimmune mice with apoptotic cells leads to the generation, albeit in low titer, of several different autoantibodies (33, 34). It should also be noted that many autoantigens on the surface of apoptotic cells are not known to be the targets of an autoimmune response.

We have recently proposed a model of immune homeostasis and tolerance in which apoptotic cells fulfill the role of self template for the immune system (13). In this role, apoptotic cells represent a renewable and continuously updated source of self Ag against which the immune system can compare all prospective Ags (foreign or self), as well as update itself as to potential changes in the definition of self (for example, during puberty or pregnancy). Indeed, recent data suggest that a dichotomy may exist in the response of T cells to APC, depending upon the source of the APC-presented Ag (35, 36, 37, 38, 39, 40). Ag derived from apoptotic cells may bias responding T cells toward anergy and tolerance, whereas Ag derived from necrotic cells, or presented in the context of activators of the innate immune system, may bias T cells toward activation and immunity (35, 36, 37, 38, 39, 40).

In terms of this model, one may envision multiple abnormalities that can predispose an individual to autoimmunity. Examples include the following: 1) errors in the generation of apoptotic self template, perhaps due to abnormalities in the executionary phase of apoptosis; 2) errors in the tagging, recognition, and/or clearance of apoptotic cells, involving either the apoptotic cell ligands or the phagocytic receptors for apoptotic cells; 3) errors in the presentation of self Ag to the immune system, perhaps due to abnormalities in processing or presentation of self Ag by phagocytic APC; and 4) errors in the reading, interpretation, and/or updating of self template in response to apoptotic self Ag.

Of these mechanisms, the one currently receiving the most attention is defective clearance of apoptotic cells (41). According to this mechanism, loss of membrane integrity in noncleared apoptotic cells leads to the release of proinflammatory cytoplasmic contents, thereby shifting the immune response from tolerance to immunity. Two recent models involving functional deletion of C1q or the Mer tyrosine kinase support this hypothesis (42, 43).

However, autoimmune diseases, such as SLE (and, perhaps, also type I DM), most likely represent syndromes, with multiple different underlying genetic abnormalities or predisposing factors leading to a common final phenotype. For example, it is conceivable that clearance of apoptotic cells may be relatively normal, but that signaling events induced by apoptotic cell clearance are in some way aberrant in autoimmune-prone individuals (18). We hypothesize that many autoimmune-prone murine strains, including NOD mice and all the major models of SLE-prone mice, may be linked by a mutation(s) affecting a common signaling element (or separate elements within a common signaling pathway) that is activated in response to apoptotic cells. This model offers an explanation for why the FBS- and apoptotic cell-dependent defect we describe affects some, but not all, M{phi}-derived cytokines. Affected cytokines would be limited to those whose LPS-stimulated induction is directly modulated by, or exhibits cross talk with, signaling pathways stimulated by the phagocytic clearance of apoptotic cells. The consequence of such aberrant signaling would be the misreading of apoptotic cells by M{phi}, and perhaps other APC, in autoimmune-prone mice. Given the critical role of apoptotic cells and the cytokine network in determining the balance between tolerance and immunity, the misreading of apoptotic cells would have broad potential to induce autoimmunity.

Immunologic abnormalities contributing to autoimmunity can operate at distinct times throughout the course of disease, from its earliest stages involving the initial loss of tolerance to later stages involving the susceptibility of specific tissues to injury and inflammation once autoantibodies have been formed. We believe that the apoptotic cell-dependent signaling abnormality, which we have characterized in this and previous publications (9, 10, 11, 12, 13), plays its most important role in the earliest stages of disease, during the initiation and/or maintenance of autoimmunity. We base this belief on several observations. Despite highly diverse genetic backgrounds, M{phi} from NOD and multiple SLE-prone strains of mice, spontaneous models of two very different autoimmune diseases, all share a common defect in cytokine regulation that is triggered by apoptotic cells. Importantly, this defect is present in mice as young as 1 wk of age (10), and shows unaltered expression throughout the life span of autoimmune mice, independent of disease and inflammatory sequelae (10, 13). Together these findings point to an early and critical role for this defect (as modeled by FBS- and apoptotic cell-dependent dysregulation of gene expression in NOD M{phi}) in the initiation and maintenance of autoimmunity. We speculate that this defect plays an important role in the breakdown of tolerance in general, and is not specific to any one organ or disease. Autoimmune diseases, such as SLE and type I DM, are polygenic diseases. Thus, it is possible that the interaction of this defect with other genes, specific to NOD mice, leads to the development of type I DM, whereas the interaction of this defect with different genes, specific to SLE-prone mice, leads to the development of systemic autoimmunity.

Although we have focused on cytokine expression, apoptotic cell-dependent abnormalities affect a much broader range of M{phi} functions. For example, when cultured in the presence of FBS or apoptotic cells, M{phi} from DM-prone NOD and SLE-prone MRL mice both showed increased adhesion to a variety of extracellular matrix proteins, whereas, upon culture in medium containing either dFBS or no FBS, adhesion of NOD and MRL M{phi} was identical with that of control M{phi} (A. Longacre, J. Koh, K.-H. Hsiao, H. Gilligan, H. Fan, and J. Levine, unpublished observations). Immunofluorescent staining of adherent NOD and MRL M{phi} cultured in the presence, but not the absence, of FBS revealed an actin-staining pattern consistent with reduced activity of Rho, a cytoplasmic G protein and cytoskeletal regulator (44, 45). Indeed, when NOD and MRL M{phi} were cultured in the presence of FBS, the percentage of active Rho was significantly decreased compared with nonautoimmune M{phi}. In sharp contrast, when NOD and MRL M{phi} were cultured in the absence of FBS, the amount of active Rho was increased to normal levels. Taken together, these results support the hypothesis that M{phi} from NOD and SLE-prone mice may have a serum- and/or apoptotic cell-dependent autoimmune phenotype that affects a broad range of M{phi} functions, including aberrant expression of multiple cytokines, increased adhesiveness to a variety of extracellular matrix proteins, and decreased activity of the cytoplasmic G protein and cytoskeletal regulator Rho.

In summary, FBS- and apoptotic cell-dependent IL-1 underexpression appears to represent a shared phenotype for murine autoimmunity. The presence of a common abnormality triggered by apoptotic cells that is present in prediseased mice from all of the major murine models of spontaneous autoimmunity tested to date, and that is absent in M{phi} from 16 nonautoimmune strains, strongly suggests that the genetic basis for this abnormality may comprise a shared background predisposing or permissive to autoimmunity.


   Acknowledgments
 
We thank Joyce Rauch for critical review of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants DK59793 (to J.S.L.) and HL69722 (to J.S.L.), and National Institutes of Health Training Grant T32DK07510 (to A.L.). Back

2 Address correspondence and reprint requests to Dr. Jerrold S. Levine, University of Chicago, Section of Nephrology, MC-5100, S-506, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: jlevine{at}medicine.bsd.uchicago.edu Back

3 Abbreviations used in this paper: DM, diabetes mellitus; CTACK, cutaneous T cell attractant chemokine; dFBS, delipidated FBS; M{phi}, macrophage; MCP, monocyte chemoattractant protein; MIP, M{phi} inflammatory protein; NOD, nonobese diabetic; NZB, New Zealand Black; NZW, New Zealand White; PI, propidium iodide; SLC, secondary lymphoid tissue chemokine; SLE, systemic lupus erythematosus. Back

Received for publication October 3, 2003. Accepted for publication February 10, 2004.


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