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Originally published online as doi:10.1189/jlb.0705408 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2006;79:155-165.)
© 2006 by Society for Leukocyte Biology

Abnormal regulation of the cytoskeletal regulator Rho typifies macrophages of the major murine models of spontaneous autoimmunity

Hanli Fan, Vimal A. Patel, Angelika Longacre and Jerrold S. Levine1

Section of Nephrology, Department of Medicine, The University of Illinois at Chicago

1 Correspondence: The University of Illinois at Chicago, Section of Nephrology, 820 South Wood Street, MC-793, Room 479/CSN, Chicago, IL 60612. E-mail: jslevine{at}uic.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages (m{phi}) from prediseased mice of all the major murine models of spontaneous autoimmunity have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. We show here that m{phi} from prediseased mice of the same models of spontaneous autoimmunity share a serum-dependent defect in the activity of Rho, a cytoplasmic G protein and cytoskeletal regulator. Affected strains include those developing lupus (BXSB, LG, MRL/l+, MRL/lpr, NZBWF1) and autoimmune diabetes (nonobese diabetic). No similar defect in Rho activity occurred in seven control strains. In the presence of serum, Rho activity in m{phi} from all autoimmune-prone strains was reduced to less than 10% of that in control mice. In contrast, under serum-free conditions, Rho activity was completely normal in autoimmune-prone m{phi}. The activities of Ras, another cytoplasmic G protein, and Rac and Cdc42, two additional G protein regulators of the cytoskeleton, were regulated normally in autoimmune-prone strains. Serum-dependent dysregulation of Rho was associated with multiple abnormalities, including increased adhesion to various surfaces, a more spread dendritic morphology, and an altered actin cytoskeletal organization. Our results suggest that m{phi} from multiple, genetically diverse, autoimmune-prone strains share a mutation or allelic difference affecting signal transduction within a specific Rho-regulatory pathway.

Key Words: rodent • lupus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the availability of several murine models of spontaneous autoimmunity, the origins of autoimmunity remain highly elusive. The reasons for this are multiple and include all of the following: the interaction of genetic and environmental factors [1 2 3 ], the potential role of stochastic factors [1 , 4 ], the polygenic nature of inherited susceptibility to autoimmunity [1 , 2 , 5 ], the existence of distinct checkpoints or stages in the development of autoimmunity, each of which may require the contribution of separate genes [1 , 2 ], and finally, the possibility that diseases such as systemic lupus erythematosus (SLE) may, like cancer, be more a syndrome than a single entity and represent a common phenotype arising from multiple, distinct causes [1 , 5 , 6 ]. Given this inordinate complexity, the existence of common cellular abnormalities, which are shared by all or many of the major models of spontaneous autoimmunity, may provide invaluable clues to the pathogenesis of autoimmunity.

We have shown that macrophages (m{phi}) from prediseased mice of all the major murine models of SLE [7 ], as well as the nonobese diabetic (NOD) strain [8 ], which spontaneously develops autoimmune diabetes mellitus (DM), have an identical defect in cytokine expression, which is triggered by serum lipids and/or apoptotic cells. Affected SLE-prone strains include MRL/+, MRL/lpr, NZB, NZW, NZBWF1, BXSB, and LG/J, which express or contribute to the development of autoimmunity [7 ]. It is important that in the absence of serum lipids and apoptotic cells, cytokine expression by m{phi} from these strains is completely normal [7 , 8 ]. This defect appears to exist only in autoimmune-prone strains, as no similar defect was found in 16 nonautoimmune control strains [7 8 9 10 11 ]. Elicitation of this defect by exposing m{phi} from prediseased autoimmune-prone mice to apoptotic cells leads to the dysregulated expression of multiple cytokines and chemokines, including interleukin-1 (IL-1), IL-6, IL-12, and tumor necrosis factor-{alpha} [7 , 8 ]. In probing the basis and consequences of this defect, we have used fetal bovine serum (FBS) as a surrogate for apoptotic cells, as lipid-containing serum fully mimics the effect of apoptotic cells in eliciting all aspects of this defect [7 , 8 ].

Recently, we showed that, at least in the case of m{phi} from SLE-prone MRL/+ and MRL/lpr mice, the consequences of this defect are not limited to dysregulated cytokine expression but also extend to the adhesive properties and cytoskeletal organization of these cells [12 ]. In the presence of FBS, MRL m{phi} adhered in increased numbers to a variety of extracellular matrix (ECM) proteins compared with m{phi} from two nonautoimmune strains. It is remarkable that, in the absence of FBS, adhesion by MRL m{phi} was similar to that by nonautoimmune m{phi}. The morphology and actin-staining pattern of adherent MRL m{phi} were consistent with the reduced activity of Rho, a cytoplasmic G protein and regulator of the cytoskeleton [13 14 15 16 ]. Indeed, MRL m{phi} cultured in the presence of FBS had markedly decreased levels of active Rho compared with nonautoimmune m{phi} [12 ]. It is remarkable that, when cultured in the absence of FBS, MRL m{phi} displayed normal Rho activity and cytoskeletal morphology [12 ]. Addition of a Rho inhibitor to normal m{phi} reproduced all the morphologic and cytoskeletal abnormalities seen in MRL m{phi} [12 ].

In the present study, we show that these FBS-dependent abnormalities in the regulation of Rho, adhesion, and cytoskeletal organization extend to prediseased mice from all the other major murine models of autoimmunity, which we have studied previously. In the presence of FBS, Rho activity in m{phi} from all autoimmune-prone strains was reduced to less than 10% of that seen in m{phi} from control, nonautoimmune mice. In marked contrast, when cultured under FBS-free conditions, Rho activity was completely normal in these autoimmune-prone m{phi}. No similar FBS-dependent dysregulation was observed for Ras, another cytoplasmic G protein. Taken together, our results suggest that m{phi} from multiple, genetically diverse, autoimmune-prone strains possess a shared autoimmune phenotype, which is triggered by FBS lipids and/or the uptake of apoptotic cells, and manifests as abnormalities within a broad range of m{phi} functions, including the expression of multiple cytokines and control of the cytoskeletal regulator Rho.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
B6.MRL-Tnfrsf6lpr (B6-lpr), B6Smn.C3-Tnfsf6gld (B6-gld), BALB/cByJ (BALB/c), BXSB/MpJ (BXSB), C3HeB/FeJ (C3HeB), C57BL/6J (C57BL/6), CBA/J (CBA), LG/J (LG), MRL/MpJ-Tnfrsf6lpr (MRL/lpr), NOD/LtJ (NOD), and NZBWF1/J (NZBWF1) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). DBA/2 (DBA) mice were purchased from Harlan Sprague-Dawley (Madison, WI). All mice were maintained under pathogen-free conditions and used between 4 and 6 weeks of age. We used only male mice, with the exception of the BXSB strain, for which we used males and females. The Institutional Animal Care and Use Committee approved all animal protocols.

M{phi} culture
Peritoneal exudate cells (PEC) were harvested by lavage, 3–4 days after intraperitoneal injection of 1.5 ml 4.05% thioglycollate broth [7 8 9 10 11 12 ]. After being washed twice in RPMI 1640, PEC were suspended in one of two media: FBS-containing R.10 culture medium (RPMI 1640 plus 10% FBS, with 2 mM L-glutamine, 5 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin) or FBS-free R.0 medium (R.10 minus FBS), supplemented with 100 U/ml human recombinant m{phi}-colony stimulating factor (hrM-CSF; generous gift of Genetics Institute, Cambridge, MA). PEC were then cultured overnight under nonadherent conditions in 100 x 20 mm tissue-culture dishes at 5–10 x 106 cells per dish. Dishes were precoated the day before addition of cells with 10 ml 1.5% agarose (Gibco, Grand Island, NY) in phosphate-buffered saline (PBS) and then equilibrated over 24 h against three changes of the same medium in which PEC were to be resuspended. After overnight culture on agarose, PEC were collected, washed in RPMI 1640, resuspended in the same medium as they had been cultured overnight, and then used for experiments. For all experiments, we used pooled m{phi} harvested from a minimum of at least five mice per strain.

3-(4,5-Dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay
The number of adherent, viable m{phi} was determined using a modification of the MTT assay [12 , 17 ], whose output is proportional to the total number of viable cells present. After washing away nonadherent cells, 165 µl MTT, diluted in R.0 (1 mg/ml), was added to each well. After incubation at 37°C for 4 h, the MTT formazan was dissolved by adding 165 µl 10% sodium dodecyl sulfate in 0.01 N HCl. Aliquots from each well were read using a microELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 650 nm. Data are presented as mean ± SE of the raw optical density (OD) values. We have previously established the linearity of this assay for cell numbers ranging from 2000 to 200,000 cells per well [12 ]. Also, we have established that graphs of MTT conversion (OD650) versus cell number have roughly equivalent slopes, irrespective of strain or the presence versus absence of FBS, thereby ensuring that mitochondrial activity per cell does not vary as a function of genetic susceptibility to autoimmunity or culture conditions [12 ].

Adhesion assay
PEC, suspended in R.10 or R.0 plus hrM-CSF (100 U/ml), were added at 1 x 105 cells per well to 96-well bacteriologic plastic (BP) plates in the presence or absence of lipopolysaccharide (LPS; 100 ng/ml). After overnight culture, wells were washed three times with RPMI 1640, and the number of remaining adherent cells was quantitated by MTT assay. For all strains of mice, adherent cells were >98% m{phi}, as confirmed by morphologic analysis and nonspecific esterase staining [7 8 9 10 11 12 ]. The percentage of added m{phi} adhering to individual wells was always >50% for those strains manifesting the greatest degree of adhesion.

Assays for cytoplasmic G proteins
After overnight, nonadherent culture in R.10 or R.0 plus hrM-CSF, 3–5 x 106 PEC were assayed for the active guanosine 5'-triphosphate (GTP)-bound forms of the four cytoplasmic G proteins, Rho, Ras, Rac, and Cdc42. The active GTP-bound form of each of these cytoplasmic G proteins constitutes <10% of its total in cells. As the active GTP-bound forms are hydrolyzed rapidly to inactive guanosine 5'-diphosphate (GDP)-bound forms, a pull-down assay was used to separate the active forms. For the Rho assay, we used a kit from Cytoskeleton (Denver, CO) containing a fusion protein between glutathione S-transferase (GST) and the Rho-binding domain of the rhotekin protein coupled to glutathione agarose beads. For the Ras assay, we used a kit from Upstate Biotechnology (Lake Placid, NY) containing a fusion protein between GST and the Ras-binding domain of Raf-1 coupled to glutathione agarose beads. For the Rac and Cdc42 assays, we used a kit from Upstate Biotechnology containing a fusion protein between GST and the Rac- and Cdc42-binding domain from p21-activated kinase (PAK)1 coupled to glutathione beads. Kit directions were followed, and all samples were maintained on ice throughout. Individual lysates, subjected to the pull-down assay (active GTP-bound forms), were then separated by polyacrylamide gel electrophoresis on a 4–20% Tris glycine gradient gel (Invitrogen, Carlsbad, CA) and transferred to a polyvinylidene difluoride membrane at 100 V for 1 h. After blocking the membrane for 4 h at room temperature in 5% nonfat milk in Tris-buffered saline/Tween 20 (TBST) buffer (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20), the membrane was incubated overnight at 4°C in TBST with a 1:800 dilution of mouse anti-RhoA immunoglobulin G1 (IgG1) monoclonal antibody (mAb; Cytoskeleton), a 1:2000 dilution of mouse anti-Ras IgG2a mAb (Upstate Biotechnology), a 1:1000 dilution of mouse anti-Rac1 IgG2b mAb (BD Biosciences, San Jose, CA), or a 1:250 dilution of mouse anti-Cdc42 IgG1 mAb (BD Biosciences) supplied with the kits. Blots were washed three times with TBST and then incubated with a 1:5000 dilution in TBST of an alkaline phosphatase-conjugated goat anti-mouse Ig, supplied as part of a detection kit and used according to the manufacturer’s instructions (Chemiluminescent Immunoblot Detection System, Applied Biosystems, Bedford, MA).

Immunofluorescence staining
After overnight, nonadherent culture, PEC were allowed to adhere to 12 mm round coverslips coated with mouse laminin (LAM)/poly-D-lysine (BD Biosciences) in R.10 or R.0 plus hrM-CSF (100 U/ml). After 16 h, nonadherent cells were washed away with PBS, and the remaining adherent m{phi} were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and then washed three times with PBS. All subsequent steps took place at room temperature. M{phi} were permeabilized with 0.1% Triton X-100 for 10 min, washed three times with PBS, and then incubated in blocking buffer (PBS containing 5% normal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100) for 1 h. M{phi} were incubated in blocking buffer containing Oregon green-conjugated phalloidin (Molecular Probes, Eugene, OR) for 1 h, washed three times with PBS, and then incubated in blocking buffer for 1 h and washed three times with PBS. Coverslips were inverted onto a glass slide with a drop of Antifade (Molecular Probes), sealed at their edges with clear nail polish, and stored overnight at 4°C for viewing the following day. Separate experiments were conducted in which m{phi} were plated at relatively low or higher density. In the presence of FBS, autoimmune m{phi} were plated in reduced numbers compared with nonautoimmune m{phi} to achieve comparable densities of adherent cells.

Microscopy
Cells were viewed under a Zeiss Axiovert microscope equipped with an Achrostigmat 32x/0.40 Ph1 objective lens and a Micromax digital camera (Integrated Microscopy Core, Cancer Center Digital Light Facility, University of Chicago, IL). Images from double or triple staining were obtained at the same plane of focus. Image capture analysis was performed using SlideBook 3.04 (Intelligent Imaging Innovations, Denver, CO), run on a Power Macintosh.

Densitometry
Densitometric analysis was performed using the UN-SCAN-IT gel digitizing software system (Silk Scientific, Orem, UT).

Statistics
Ten replicate wells were examined in each experiment, and the results were averaged. A minimum of three experiments was performed for all data points. Data are expressed as mean ± SEM of the averaged values obtained from each experiment. Statistical significance was determined by a two-tailed Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
FBS-dependent increased adhesiveness is present in m{phi} from all the major murine models of spontaneous autoimmunity
M{phi} from prediseased mice of the SLE-prone strains, MRL/+ and MRL/lpr, adhere in increased numbers to a variety of surfaces and ECM proteins as compared with m{phi} from the nonautoimmune control strains, BALB/c and C57BL/6 [12 ]. To determine whether FBS-dependent abnormalities of adhesion extend to prediseased mice from the other major murine models of spontaneous autoimmunity, we used a MTT-based adhesion assay, in which the relative number of m{phi} that remained adherent after vigorous washing was assessed by mitochondrial conversion of pale-yellow MTT to dark-blue crystals of MTT formazan [12 , 17 ].

As shown in Figure 1 , increased adhesion to BP segregates with genetic susceptibility to autoimmunity. When cultured in the presence of FBS, m{phi} from six autoimmune-prone strains (male BXSB, female BSXB, LG, MRL/lpr, NOD, and NZBWF1) adhered in increased numbers to BP, as compared with seven nonautoimmune control strains (BALB/c, B6-gld, B6-lpr, C57BL/6, C3HeB, CBA, and DBA). The relative increase in the number of adhering m{phi} from autoimmune-prone strains, as compared with control, nonautoimmune-prone strains, was approximately twofold (0.47±0.01 vs. 0.23±0.02, P<2x10–6). It is important that all the major SLE-prone strains as well as the NOD strain, which spontaneously develops autoimmune diabetes mellitus, showed similar behavior. These data suggest that FBS-dependent, increased adhesiveness may be a generalized feature of autoimmunity.



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  		Figure 1. M{phi} from autoimmune-prone strains adhere in increased numbers in the presence but not the absence of serum (FBS). After overnight, nonadherent culture, peritoneal m{phi} from the indicated strains were plated onto BP in the presence (R.10) or the absence (R.0) of 10% FBS, with or without LPS (100 ng/ml). After 16 h, nonadherent m{phi} were washed away, and the relative number of remaining adherent m{phi} was quantitated by MTT assay. In the presence of FBS, all pair-wise comparisons between autoimmune-prone [MRL/lpr, NOD, NZBWF1, LG, BXSB male (M), or BSXB female (F)] and control nonautoimmune (B6-lpr, B6-gld, C57BL/6, BALB/c, CBA, DBA, or C3HeB) m{phi} were statistically significant (P<0.001), irrespective of the presence of LPS. Data are combined from n = 3 independent experiments.

 
To rule out the possibility that subclinical disease or inflammation may have contributed to the observed differences, we repeated these studies in the presence of LPS (100 ng/ml), an extremely potent activator of m{phi}, as a means of achieving a similar degree of activation across all m{phi}. As seen in Figure 1 , LPS had essentially no effect on the pattern of enhanced adhesion seen with autoimmune-prone versus nonautoimmune control m{phi} (0.48±0.02 vs. 0.20±0.02, P<3x10–6). Two additional aspects of our data argue against a contribution of disease. To minimize any effects of disease or inflammation, we used only young, prediseased, autoimmune-prone mice, less than 6 weeks of age, as a source of m{phi}. Moreover, when we compared m{phi} from prediseased (<6 weeks) versus overtly diseased (>6 months) MRL/lpr or NOD mice, the overall pattern and magnitude of increased adhesion did not differ (ref. [12 ] and not shown).

It is remarkable that when cultured under FBS-free conditions, m{phi} from all six autoimmune-prone strains no longer adhered in increased numbers (Fig. 1) . Equivalent adhesion was independent of the state of m{phi} activation, occurring in the absence and the presence of LPS [no LPS: 0.17±0.02 vs. 0.22±0.02, P=not significant (NS); plus LPS: 0.15±0.02 vs. 0.22±0.02, P=NS]. Thus, m{phi} from diverse, autoimmune-prone backgrounds share a common abnormality in adhesiveness, which is triggered by FBS. These data complement our earlier studies showing that m{phi} from these SLE-prone strains and from the autoimmune diabetes-prone NOD strain have a FBS-dependent abnormality in the expression of multiple cytokines [7 , 8 ].

The genetic basis for this defect appears to reside in the autoimmune background and is independent of such genes as lpr or gld, which exacerbate and accelerate the course of autoimmunity but are themselves weak inducers of autoimmunity [18 ]. Thus, not only was the pattern and magnitude of increased adhesiveness indistinguishable in MRL/+ versus MRL/lpr mice, but also, no abnormality of adhesion was found in m{phi} from C57BL/6 mice homozygous for lpr or gld. Moreover, FBS-dependent abnormalities of adhesion were also independent of the Y chromosome-linked, accelerant gene Yaa [19 ], as male and female SLE-prone BXSB mice had similar defects. Finally, background susceptibility to autoimmunity appears to be the major determinant of this abnormality, as no consistent statistically significant differences in relative adhesion were seen among individual autoimmune-prone m{phi} or among individual nonautoimmune control m{phi}.

Although the results depicted in Figure 1 were obtained after 24 h of adhesion, an identical pattern of increased adhesion by autoimmune-prone m{phi} was observed after 1 h and 4 h of adhesion (not shown). It should be stressed that elicited peritoneal m{phi} are terminally differentiated and nonproliferative [20 , 21 ], so that these results solely reflect differences in adhesion. FBS-dependent, increased adhesion was equally manifest when LAM was substituted for BP (not shown). Thus, as previously shown with MRL m{phi} [12 ], FBS-dependent, increased adhesion by autoimmune-prone m{phi} cannot be attributed to abnormalities of a single adhesion receptor.

FBS-dependent changes in morphology and organization of the actin cytoskeleton are present in m{phi} from all the major murine models of spontaneous autoimmunity
FBS-dependent, increased adhesion by MRL m{phi} is associated with several notable changes in morphology and actin cytoskeletal organization [12 ]. We therefore determined whether FBS-dependent morphologic and cytoskeletal changes also occur in m{phi} from the other autoimmune-prone strains.

After overnight, nonadherent culture, m{phi} were allowed to adhere to LAM or BP in the presence or absence of FBS. It is remarkable that m{phi} from all autoimmune-prone strains demonstrated identical FBS-dependent changes in their morphology and actin cytoskeletal organization, as compared with m{phi} from control, nonautoimmune strains (Figs. 2 and 3 ). Differences between autoimmune-prone and control m{phi} were evident only when m{phi} were cultured in the presence of FBS. When cultured in the absence of FBS, autoimmune-prone and control m{phi} were indistinguishable.



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  		Figure 2. M{phi} from autoimmune-prone mice have FBS-dependent changes in morphology and actin cytoskeletal organization. After overnight, nonadherent culture, peritoneal m{phi} from BALB/c and MRL/lpr mice were plated onto LAM-coated coverslips in the presence (FBS) or absence (FBS-free) of FBS. After 16 h, m{phi} were examined by epifluorescence microscopy for staining of filamentous F-actin. In the presence of FBS, BALB/c m{phi} were uniformly round, whereas MRL/lpr m{phi} had a more spread, often elongated "dendritic" morphology (indicated by white arrowheads). The apparent increase in the size of FBS-cultured MRL/lpr m{phi} as compared with BALB/c m{phi} reflects greater spreading and not a difference in cell volume. The overall intensity of actin staining was diminished consistently in MRL/lpr compared with BALB/c m{phi} (FBS [unadj]). To highlight the FBS-dependent alterations in cytoskeletal organization common to m{phi} from all autoimmune-prone strains, we increased the exposure time so that the average fluorescent intensity for images of MRL/lpr versus BALB/c m{phi} was equivalent (FBS [adj]). BALB/c m{phi} usually contained a subcortical actin ring (indicated by yellow arrows). MRL/lpr m{phi}, conversely, in the presence of FBS, consistently lacked this subcortical actin ring and, instead, frequently displayed a concentrated staining of subcortical actin within dense, plaque-like structures near the cell periphery (indicated by white arrows). In the absence of FBS (FBS-free [unadj]), MRL/lpr and BALB/c m{phi} were indistinguishable from each other and contained a subcortical actin ring (indicated by yellow arrows). All panels were photographed at an equivalent original magnification (40x) and scaled to the same size. The insets in each panel show a typical single cell. All insets represent an additional 3x original magnification, with the exception of MRL/lpr for FBS [unadj] and FBS [adj], which were originally magnified only 2x, because of the greater spreading and increased apparent size of MRL/lpr m{phi} in the presence of FBS. Shown are representative photographs from n = 3 independent experiments.

 


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  		Figure 3. FBS-dependent abnormalities in actin cytoskeletal organization occur in m{phi} from all the major models of spontaneous autoimmunity. After overnight, nonadherent culture, peritoneal m{phi} from (A) control, nonautoimmune and (B) autoimmune-prone strains were plated onto LAM-coated coverslips in the presence (FBS) versus absence (FBS-free) of FBS. After 16 h, m{phi} were examined by epifluorescence microscopy for filamentous F-actin staining. (A) M{phi} from control strains were uniformly round, consistently contained a subcortical actin ring, and did not differ in their staining properties when cultured in the presence versus absence of FBS. (a) BALB/c; (b) C3HeB; (c) C57BL/6; (d) B6-gld; (e) B6-lpr; (f) CBA; (g) DBA. (B) Autoimmune-prone m{phi} differed markedly in their actin cytoskeletal organization depending on the presence versus absence of FBS during culture. In the absence of FBS, autoimmune-prone m{phi} closely resembled control m{phi} in that they were round and consistently contained a subcortical ring (indicated by yellow arrows). However, in the presence of FBS, autoimmune-prone m{phi} consistently lacked a subcortical actin ring. Instead, they frequently assumed a more spread dendritic morphology (indicated by white arrowheads) and displayed a concentrated staining of subcortical actin within dense, plaque-like structures near the cell periphery (indicated by white arrows). It should be noted that the overall intensity of actin staining was markedly diminished in FBS-cultured, autoimmune-prone m{phi}. For this reason, the exposure times for all micrographs of FBS-cultured, autoimmune-prone m{phi} were increased, so that the average fluorescent intensity of these images was comparable with that of m{phi} from control strains. Without this adjustment, the majority of FBS-cultured, autoimmune-prone m{phi} was largely invisible. The diminished intensity in unadjusted versus adjusted fluorescent images, as shown in Figure 2 , is representative of m{phi} from all autoimmune-prone strains. (a) Female BXSB; (b) male BXSB; (c) LG; (d) MRL/lpr; (e) NOD; (f) NZBWF1. All panels were photographed at an equivalent original magnification (40x) and scaled to the same size. Shown are representative photographs from n = 2 independent experiments.

 
These FBS-dependent alterations may be summarized as follows. First, autoimmune-prone m{phi} assumed a more spread morphology as compared with control, nonautoimmune m{phi}, occupying, on average, a larger surface area per cell. It is important to stress that all panels within each individual figure (Figs. 2 and 3) were photographed at equal magnifications. The apparent increase in size of autoimmune m{phi}, when cultured in the presence of FBS, reflects increased spreading and consequent occupying of a larger surface area. Autoimmune-prone and control m{phi} in suspension, irrespective of whether they are cultured in the presence or absence of FBS, are of the same size and have the same diameter, as assessed by forward scatter on flow cytometry (ref. [12 ] and not shown). In addition to increased spreading, autoimmune m{phi} often assumed a more elongated, dendritic morphology, with long cytoplasmic extensions (Figs. 2 and 3 , white arrowheads). In marked contrast, under FBS-free culture, autoimmune m{phi} were less spread and more rounded, closely resembling control, nonautoimmune m{phi}. The spreading and morphology of control, nonautoimmune m{phi} were similar, irrespective of the presence or absence of FBS, and resembled autoimmune-prone m{phi} cultured under FBS-free conditions.

Second, in accord with the increased adhesion shown by autoimmune m{phi}, we observed dramatic FBS-dependent differences in the level of polymerized F-actin. Phalloidin binds to polymerized, filamentous F-actin but not to unpolymerized G-actin. In the presence of FBS, the overall intensity of F-actin staining was diminished consistently in autoimmune-prone compared with control, non-autoimmune m{phi}. This difference in the intensity of actin staining is shown for MRL/lpr versus BALB/c m{phi} (Fig. 2 , FBS [unadj]). A similar, striking decrease in the intensity of actin staining was observed for m{phi} from all six autoimmune-prone strains (Fig. 3 , and not shown). To make apparent the FBS-dependent alterations in cytoskeletal organization, common to m{phi} from all autoimmune-prone strains, we increased the exposure time so that the average fluorescent intensity for images of autoimmune-prone versus control m{phi} was equivalent (Fig. 3) . Without this adjustment, the majority of autoimmune-prone m{phi} was all but invisible when stained for filamentous F-actin. A representative example of the effects of this adjustment is shown for MRL/lpr m{phi} (Fig. 2) . In viewing the actin-staining patterns for m{phi} from the other autoimmune-prone strains (Fig. 3) , the marked decrement in actin staining for autoimmune m{phi}, when cultured in the presence of FBS, should be borne in mind.

Finally, autoimmune and nonautoimmune m{phi} also demonstrated FBS-dependent differences in the organization of their actin cytoskeleton. M{phi} from nonautoimmune mice, irrespective of the presence or absence of FBS, usually contained a subcortical actin ring (yellow arrows), most visible in a higher plane of focus (Figs. 2 and 3) . In marked contrast, autoimmune-prone m{phi}, when cultured in the presence of FBS, consistently lacked this subcortical actin ring (Figs. 2 and 3) . Instead of a subcortical actin ring, autoimmune-prone m{phi} frequently displayed a concentrated staining of subcortical actin within dense, plaque-like structures near the cell periphery (white arrows). It is notable that under FBS-free conditions, the pattern of actin staining for autoimmune-prone m{phi} did not differ from that of control, nonautoimmune m{phi} and closely resembled that seen in FBS-cultured, control, nonautoimmune m{phi} (Figs. 2 and 3) .

All of these FBS-dependent differences, including increased spreading, decreased filamentous F-actin, and altered cytoskeletal organization, were also seen when autoimmune-prone m{phi} were cultured on BP and irrespective of whether autoimmune-prone m{phi} were cultured under conditions of high or low density (ref. [12 ] and not shown).

FBS-dependent dysregulation of Rho, but not Ras, Rac, and Cdc42, occurs in m{phi} from all the major murine models of spontaneous autoimmunity
The observed FBS-dependent abnormalities observed in m{phi} from autoimmune-prone mice, including increased adhesion, increased spreading, decreased F-actin staining, and altered cytoskeletal organization, are consistent with reduced activity of the cytoskeletal regulator and G protein Rho [13 14 15 16 ]. To determine whether FBS-dependent dysregulation of Rho activity may underlie these abnormalities in adhesion, we measured Rho activity directly.

After overnight, nonadherent culture in the presence or absence of FBS, lysates from an equal number of m{phi} were examined for active Rho·GTP, according to assay specifications [22 ]. When cultured under FBS-free conditions, the levels of active Rho·GTP did not differ among m{phi} from autoimmune-prone versus control, nonautoimmune mice (Fig. 4 ). In contrast, in the presence of FBS, the level of active Rho·GTP was reduced sharply in autoimmune-prone versus nonautoimmune m{phi} (Fig. 4) . To compare our data quantitatively, we normalized all Rho·GTP activity to that seen in BALB/c m{phi} cultured under FBS-containing conditions (set at 100%). In the absence of FBS, there was no significant difference in Rho·GTP between autoimmune-prone and nonautoimmune m{phi} (116.7±12.6% vs. 141.4±16.4%, P=NS; Fig. 5A ). In marked contrast, when m{phi} were cultured in the presence of FBS, the level of Rho·GTP was reduced severely in autoimmune-prone m{phi} (1.8±0.7% vs. 134.9±13.5%, P<2x10–6; Fig. 5B ).



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  		Figure 4. FBS-dependent dysregulation of Rho, but not Ras, occurs in m{phi} from all the major murine models of spontaneous autoimmunity. After overnight, nonadherent culture in the presence (+FBS) or absence (–FBS) of FBS, lysates prepared from equal numbers of m{phi} from the indicated strains were examined for active Rho or Ras by a pull-down assay using a fusion protein between GST and the Rho-binding domain of the rhotekin protein or the Ras-binding domain of Raf-1, respectively. When m{phi} were cultured in the presence of FBS, the quantity of active Rho·GTP was reduced in autoimmune-prone m{phi} compared with control, nonautoimmune m{phi}. Under FBS-free conditions, the quantity of active Rho·GTP in autoimmune-prone m{phi} was normalized to levels seen in control m{phi} and exceeded that found in FBS-cultured, autoimmune-prone m{phi}. No differences in the quantity of active Ras·GTP were seen between autoimmune-prone and control m{phi}, irrespective of the presence or absence of FBS. To ensure that lysates used in the pull-down assay were derived from equivalent numbers of m{phi}, an aliquot of the original lysate (before mixing with agarose beads in the pull-down assay) was probed for total Rho or total Ras (not shown). Shown are representative blots from n = 3 independent experiments.

 


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  		Figure 5. FBS-dependent dysregulation in the activity of Rho, but not Ras, is a characteristic of m{phi} from all the major autoimmune-prone strains. After overnight, nonadherent culture in the presence (FBS) or absence (FBS-free) of FBS, lysates from equal numbers of m{phi} from the indicated strains were examined for active Rho·GTP or Ras·GTP. Relative levels of Rho·GTP and Ras·GTP were determined by densitometric scanning for each strain and normalized for total Rho and Ras, respectively. Relative levels of Rho·GTP are plotted for (A) FBS-free and (B) FBS-containing conditions. (C) In addition, for each strain, we determined the ratio of activity of Rho and Ras under FBS-containing versus FBS-free conditions (FBS:FBS-free) and compared these ratios for autoimmune-prone versus nonautoimmune, normal strains. These data are representative of n = 3 independent experiments.

 
To confirm that the FBS-dependent decrease in Rho·GTP observed in m{phi} from autoimmune-prone mice is a specific signaling defect and not a generalized defect of cytoplasmic G proteins, we also determined the activity of Ras. No differences in the level of active Ras·GTP could be detected between autoimmune-prone and nonautoimmune m{phi} in the presence or absence of FBS (Fig. 4) . Thus, when we compared the ratio of Ras activity in the presence of FBS with that in the absence of FBS (RasFBS/RasFBS-free), there was no difference in this ratio for autoimmune-prone versus control m{phi} (109.8±16.4% vs. 101.4±10.9%, P=NS; Fig. 5C ). In marked contrast, the ratio of Rho activity in the presence of FBS to that in the absence of FBS (RhoFBS/RhoFBS-free) was reduced significantly for autoimmune-prone versus control m{phi} (1.6±0.8% vs. 97.6±5.7%, P<10–8; Fig. 5C ).

In addition to Rho, two other cytoplasmic G proteins play a prominent role in the regulation of the cytoskeleton, Rac, and Cdc42. To confirm that the FBS-dependent decrease in Rho·GTP is specific to Rho and not a generalized abnormality of cytoskeletal regulators, we determined the activity of Rac and Cdc42 in m{phi} from selected autoimmune-prone and nonautoimmune control strains. No differences in the level of active Rac·GTP or Cdc42·GTP could be detected between autoimmune-prone and nonautoimmune m{phi} in the presence or absence of FBS (Fig. 6 ). These data demonstrate that m{phi} from multiple autoimmune-prone strains appear to have a specific FBS-dependent defect in the regulation of Rho.



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  		Figure 6. M{phi} from mice developing spontaneous autoimmunity show no FBS-dependent abnormalities in the regulation of the activity of Rac and Cdc42. After overnight, nonadherent culture in the presence (+FBS) or absence (–FBS) of FBS, lysates prepared from equal numbers of m{phi} from the indicated strains were examined for active Rac and Cdc42 by a pull-down assay using a fusion protein between GST and the Rac- and Cdc42-binding domain of PAK1. No differences in the quantities of active Rac·GTP or active Cdc42·GTP were seen between autoimmune-prone and control m{phi}, irrespective of the presence or absence of FBS. To ensure that lysates used in the pull-down assay were derived from equivalent numbers of m{phi}, an aliquot of the original lysate (before mixing with agarose beads in the pull-down assay) was probed for total Rac or total Cdc42 (not shown). Shown are representative blots from n = 2 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We show here that m{phi} from prediseased mice of all the major murine models of spontaneous autoimmunity share a FBS-dependent abnormality in the activity of Rho, a cytoplasmic G protein and major regulator of the cytoskeleton [13 14 15 16 ]. Affected strains include those developing SLE (BXSB, LG, MRL/+, MRL/lpr, NZBWF1) and autoimmune DM (NOD). We found no similar defect in Rho activity in seven nonautoimmune, control strains. FBS-dependent dysregulation of Rho activity was associated with a number of adhesive abnormalities in m{phi} from autoimmune-prone mice. In the presence of FBS, m{phi} from autoimmune-prone mice adhered in increased numbers to a variety of surfaces and ECM proteins. In addition, under microscopic examination, they demonstrated an altered cytoskeletal organization, leading to a more spread dendritic morphology and an altered actin-staining pattern, consistent with reduced activity of Rho. In marked contrast, under FBS-free conditions, autoimmune-prone m{phi} were indistinguishable in all respects from control, nonautoimmune m{phi}.

Although our data do not definitively establish a causal relationship between reduced Rho activity and these adhesive and cytoskeletal abnormalities, it is noteworthy that inhibition of Rho with Clostridium botulinum C3 exoenzyme [23 ] in nonautoimmune m{phi} reproduced the morphologic and cytoskeletal abnormalities seen in autoimmune-prone m{phi} [12 ]. Moreover, the functional, morphologic, and cytoskeletal abnormalities demonstrated by autoimmune-prone m{phi} cultured in the presence of FBS are characteristic of m{phi} having reduced or absent Rho activity, produced by pharmacologic or molecular biologic means [24 25 26 27 28 29 30 ]. Thus, inhibition of Rho in normal m{phi} leads to all of the following: increased adhesiveness [26 , 28 , 29 ], greater spreading with assumption of an elongated dendritic morphology [24 , 25 , 28 , 29 ], decreased F-actin [24 , 25 ], and cytoskeletal reorganization with loss of the subcortical actin ring and appearance of dense, plaque-like actin structures near the cell periphery [24 , 25 ]. In understanding these results, it is crucial to note that the cytoskeletal organization of m{phi} and other white blood cells differs in several important ways from that of fibroblasts, the cell most commonly used to study the effect of cytoskeletal organization on morphology and adhesion. As opposed to fibroblasts, cultured m{phi} do not form focal adhesion complexes [13 14 15 16 ]. We verified the absence of focal adhesion complexes in control and autoimmune-prone m{phi} by staining for the focal adhesion complex protein vinculin (ref. [12 ] and not shown). In keeping with the absence of focal adhesion complexes, Rho-activated m{phi} also do not assemble actin into polymerized bundles of stress fibers, which, in fibroblasts, converge on focal adhesion complexes. Instead, Rho-activated m{phi} possess a finer contractile network of actin filaments [13 14 15 16 ]. Without the anchoring provided by focal adhesion complexes, Rho-activated contraction of this actin filament network in m{phi} leads to rounding up of m{phi}, as opposed to the spread out morphology seen in Rho-activated fibroblasts [13 14 15 16 ]. Presumably, the diminished Rho activity seen in autoimmune-prone m{phi}, cultured in the presence of FBS, results in less contraction of the actin network, permitting the cell to spread and adhere more.

Rho is a member of the Ras superfamily of small, GTP-binding proteins [13 14 15 16 ]. Members of this superfamily cycle between an active GTP-bound state and an inactive GDP-bound state, thereby functioning as molecular switches or timers. Rho is active only as long as it remains in a GTP-bound form. Upon hydrolysis of GTP to GDP, Rho loses its activity. In its active, GTP-bound form, Rho interacts with effector or target molecules to initiate downstream responses. The proportion of active Rho is determined by two major reactions. GTPase-activating proteins (GAP) augment the low, intrinsic GTPase activity of Ras superfamily members and therefore decrease the proportion of active Rho. Guanine nucleotide exchange factors (GNEF) catalyze the dissociation of GDP from Ras superfamily members. As the intracellular concentration of GTP is much greater than that of GDP, the spontaneous reassociation of Rho with guanine nucleotides leads to an increased proportion of active Rho. An additional level of regulation is provided by guanine nucleotide dissociation inhibitors (GDI), which sequester Rho in the cytoplasm and prevent Rho from interacting with other membrane-associated signaling molecules.

Regulation of Rho activity is extremely complex, with over 10 GAP, over 30 GNEF, and four GDI identified to date [13 14 15 16 ]. As Rho activity in autoimmune-prone m{phi} is normalized under FBS-free conditions, it is unlikely that the Rho gene itself is mutated in these mice. Rather, the responsible abnormality most likely involves one or more of the GAP, GNEF, and GDI, which interact directly with Rho or other pathways further upstream, regulating these GAP, GNEF, and GDI.

In understanding the significance of this abnormality, it is important to recognize that the consequences of dysregulated Rho activity may not be limited to the adhesive properties of autoimmune-prone m{phi}. The number of downstream targets and effector functions known to be influenced by Rho is increasing continually [14 , 15 ]. Many of these are dependent on the actin cytoskeleton and include cell migration [27 28 29 ], complement-dependent phagocytosis [31 ], and cytokinesis [15 ]. It is important that activated Rho also regulates a number of other signal transduction pathways that are independent of the cytoskeleton. These include activation of the transcription factors serum response factor [32 ] and nuclear factor-{kappa}B [33 ], cell cycle progression [22 ], and cadherin-mediated cell–cell contact [34 ]. Hence, an abnormality in the regulation of Rho could have wide-reaching effects on immune homeostasis, affecting such m{phi} functions as migration, trafficking, antigen uptake, and interaction with other immune cells. All of these functions are crucial to the balance between tolerance and immunity. Indeed, alterations in the function or expression of various adhesion molecules have been shown to modulate the course of disease in several murine models of autoimmunity [35 36 37 38 39 40 ].

Recent studies have shown that cytoskeletal events within the antigen-presenting cell (APC) play a critical role in formation of the immunologic synapse and T cell activation [41 , 42 ]. Therefore, the Rho-dependent, cytoskeletal abnormalities that we have described in autoimmune-prone m{phi} may affect the manner in which T cells are activated. Given that dysregulated Rho activity is dependent on the presence of FBS (a surrogate for apoptotic cells), abnormalities in T cell activation may be limited to those antigens that are presented by APC in the context of apoptotic cells. The potential dependence of dysregulated Rho activity on exposure to apoptotic cells is especially noteworthy in that we and others have shown that apoptotic cells and their products are the antigenic targets of multiple autoantibodies across a broad spectrum of autoimmune diseases, including SLE, antiphospholipid syndrome, scleroderma, and antineutrophil cytoplasmic antibody-positive vasculitis [43 44 45 46 47 48 49 50 ].

In summary, we have shown that m{phi} from multiple SLE-prone strains, as well as the autoimmune diabetes-prone NOD strain, share a FBS-dependent abnormality in the regulation of Rho activity. Coupled with our earlier work on FBS- and apoptotic cell-dependent cytokine dysregulation in these same autoimmune-prone strains [7 , 8 ], our results suggest that m{phi} from all the major autoimmune-prone strains have a shared autoimmune phenotype, which is triggered by FBS and/or the uptake of apoptotic cells, and manifest as abnormalities within a broad range of m{phi} functions. In the absence of FBS or apoptotic cells, m{phi} from autoimmune-prone mice behave normally. We were not able to establish a concordance between FBS and apoptotic cells in terms of eliciting dysregulated activity of Rho, as we measured Rho activity in nonadherent m{phi} immediately prior to adhesion, and nonadherent m{phi} failed to interact with apoptotic cells. We hypothesize that apoptotic cells will mimic the effects of FBS on Rho in adherent m{phi} and that autoimmune-prone mice share a mutation or allelic difference affecting signal transduction within a specific Rho-regulatory pathway, which is triggered by FBS lipids and/or the recognition and uptake of apoptotic cells.

The consequence of such aberrant signaling of regulatory molecules such as Rho would be the "misreading" of apoptotic cells by m{phi} and perhaps other APC in autoimmune-prone mice. Many current models of autoimmunity suggest that delayed or reduced clearance of apoptotic cells is the critical event leading to the emergence of autoimmunity [51 ]. According to these models, delayed clearance of apoptotic cells leads to the loss of cell membrane integrity, leakage of intracellular contents, and the presentation of apoptotic antigens in the context of inflammatory signals with resultant autoimmunity. Indeed, targeted deletion of several molecules involved in the clearance of apoptotic cells (e.g., C1q, MER, and MFG-E8) leads to autoimmunity and impaired apoptotic cell clearance [52 53 54 ]. However, delayed clearance is not the sole consequence of these targeted deletions. An overlooked consequence is that a reduction in the clearance of apoptotic cells necessarily implies a reduction in the signal transduction events induced in phagocytic cells using those targeted molecules for recognition and/or uptake of apoptotic cells. As shown by us and others [21 , 55 ], the recognition and/or uptake of apoptotic cells are accompanied by intracellular signal transduction. In contradiction to the delayed clearance model, we have recently found that mitogen-activated protein kinase (MAPK) signaling events induced in m{phi} from nonautoimmune control mice by late apoptotic cells that have lost membrane integrity were identical to those induced by early apoptotic cells with intact cell membranes (V. A. Patel et al., manuscript submitted). Moreover, the MAPK signaling events induced by early and late apoptotic cells were strongly dominant over those induced by necrotic cells [56 ]. These results are inconsistent with the delayed clearance model and imply that regardless of membrane integrity, apoptotic cells have a uniform effect on m{phi} signal transduction, at least with respect to MAPK pathways. We therefore propose that the absence of apoptotic cell-induced signaling events, rather than delayed apoptotic cell clearance, is the critical event leading to the loss of tolerance and the development of autoimmunity. The results reported here are consistent with our theory that abnormalities in the signaling events induced by apoptotic cells may comprise a permissive or predisposing background to development of autoimmunity.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants DK59793 and HL69722 and by a Young Investigator Award from the National Kidney Foundation of Illinois. The authors thank the University of Chicago Integrated Microscopy Core, Cancer Center Digital Light Facility, especially Shirley Bond, for assistance in microscopic analysis and Joyce Rauch, Martin A. Schwartz, and David S. Ucker for critical review of the manuscript.

Received July 23, 2005; revised September 5, 2005; accepted September 6, 2005.


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