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Blood, 1 February 2007, Vol. 109, No. 3, pp. 1248-1256. Prepublished online as a Blood First Edition Paper on October 3, 2006; DOI 10.1182/blood-2006-03-012898.
PHAGOCYTES Dual role for RhoA in suppression and induction of cytokines in the human neutrophil1 Department of Medicine, National Jewish Medical and Research Center, Denver, CO; 2 Department of Medicine, University of Colorado School of Medicine, Denver; 3 Department of Toxicology, Hannover Medical School, Hannover, Germany; 4 Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO
Production of tumor necrosis factor-  (TNF) by the neutrophil (PMN) is a pivotal event in innate immunity, but the signals regulating TNF induction in this primary cell are poorly understood. Herein, we use protein transduction to identify novel, opposing anti– and pro–cytokine-inducing roles for RhoA in the resting and lipopolysaccharide (LPS)–stimulated human PMN, respectively. In the resting cell, RhoA suppresses Cdc42 activation, I B degradation, nuclear factor-B (NF-B) activation, and induction of TNF and NF-B–dependent chemokines. Suppression of TNF induction by RhoA is Rho kinase (ROCK) independent, but Cdc42 dependent, because TNF induction by C3 transferase is attenuated by inhibition of Cdc42, and constitutively active Cdc42 suffices to activate NF-B and induce TNF. By contrast, we also place RhoA downstream of p38 mitogen-activated protein kinase and Cdc42 in a novel LPS-activated pathway in which p38, Cdc42, and ROCK all promote TNF protein expression. The p65 subunit of NF-B coprecipitates with RhoA in a manner sensitive to the RhoA activation state. Our findings suggest a new, 2-faced role for RhoA as a checkpoint in innate immunity.
The prototypical early-response cytokine TNF has multiple regulatory effects on both inflammation and host defense. Depending on the context, "positive" effects of TNF, such as enhanced bacterial killing and polymorphonuclear leukocyte (PMN) recruitment to sites of infection, may be overshadowed by negative consequences for the organism, such as aggravated organ injury.1,2 Likely, because of these mixed effects of TNF, induction of TNF by LPS and other stimuli is precisely regulated at multiple steps, including transcription, mRNA stability, translation, and protein stability.3–5 Further complicating this issue, TNF produced by different cell types has been described to have distinct and nonredundant functions in vivo.6 Moreover, multiple examples exist of cell-type–dependent differences in the pathways regulating TNF expression.5,7,8 Hence, findings derived from cell lines and even primary cells may not be applied universally, and, moreover, systemic pharmacotherapies aiming to modulate TNF production are likely to be confounded by important differences among target tissues.
The human PMN is a pivotal acute-response effector cell in inflammation and host defense and an important source of TNF
The Rho GTPases, of which the best-described members include RhoA, Cdc42, and Rac1/2, are molecular switches that have classically been associated with chemotaxis, superoxide anion (O
Reagents and antibodies
Endotoxin-free reagents and plastics were used throughout. Aprotinin, leupeptin, AEBSF, NaF, Na3VO4, DMSO, and protein A–Sepharose were from Sigma (St Louis, MO), and SB203580 was from Calbiochem (San Diego, CA). RhoA and Cdc42 assay kits and glutathione-S-transferase (GST) were from Upstate Cell Signaling Solutions (Lake Placid, NY). C3 transferase, Rhotekin-RBD-GST, L61Cdc42, GST-wt RhoA, GST-L63RhoA, and GST-Sepharose were from Cytoskeleton (Denver, CO). pET23–wild-type Wiskott-Aldrich syndrome protein (WASP)–CRIB (amino acids 201-321) and mutant non–Cdc42-binding WASP-CRIB (F271C, H246D, H249D) constructs were kind gifts from Klaus Hahn (University of North Carolina) and were expressed as C-terminal His6-tagged fusion proteins in Escherichia coli, purified, and quantified by Bradford assay.25 E coli 0111:B4 LPS was from List Biological Laboratories (Campbell, CA). Antibodies include rabbit anti-RhoA (Upstate, Lake Placid, NY), -ROCK PMN isolation and treatments PMNs were isolated from whole blood of healthy donors by discontinuous plasma Percoll centrifugation, as reported.8 For LPS exposure experiments (100 ng/mL), the cells were resuspended in RPMI 1640 culture medium (BioWhittaker, Walkersville, MD) supplemented with 10 mM HEPES (pH 7.6) and 1% human heat-inactivated platelet-poor plasma. Approval was obtained from the National Jewish Medical and Research Center Institutional Review Board for the studies. Informed consent was provided according to the Declaration of Helsinki. RhoA and Cdc42 activation assays RhoA and Cdc42 activation were assayed as described.26,27 In vitro kinase assays
A modification of a published protocol for assaying ROCK kinase was used.28 Briefly, precleared lysates were immunoprecipitated with 1.5 µg rabbit anti-ROCK Protein transduction Recombinant proteins were transduced into PMNs using a modification of a published protocol.24 Briefly, 2 µL BioPORTER in methanol was divided into aliquots into 1.5-mL reaction tubes and dried overnight. Varying quantities (4-20 µg) of recombinant proteins plus PBS, pH 7.4, up to a final volume of 40 µL were used to rehydrate the BioPORTER per the manufacturer's protocol, whereupon 5 x 106 PMNs in Krebs Ringer Phosphate Dextrose buffer were added to a final volume of 1 mL. PMNs were then incubated (2-4 hours, 25°C), pelleted (500g, 3 minutes), and resuspended. PMNs were treated with C3 transferase (20 µg/mL, 4 hours, 37°C, 107 cells/mL), a modification of a previous report.29 PCR and ELISA
RNA was isolated using Trizol reagent per the manufacturer's protocol, and reverse transcription–polymerase chain reaction (RT-PCR) was performed as previously reported.8 PMN supernatants were analyzed by ELISA for TNF Nuclear/cytoplasmic fractionation The NE-PER kit (Pierce) was used, per the manufacturer's protocol. Rhodamine-phalloidin staining and microscopy Rhodamine-phalloidin staining and microscopy were performed as previously reported.30 Briefly, PMNs were fixed with an equal volume of 4% paraformaldehyde, 3% sucrose/PBS (30 min, 37°C), settled on coverslips, permeabilized with 0.02% Tween 20/PBS (5 min, room temperature), washed once, stained with 5 units of rhodamine-phalloidin (Molecular Probes, Eugene, OR), and then mounted with Gel/Mount (Biomeda, Foster City, CA). Photographs were taken using a Zeiss Axiovert 200M microscope (Thornwood, NY) equipped with a 60x/1.40 numerical aperture oil objective and a Cooke Sensicam 2.0 camera (Cooke, Romulus, MI). Images were acquired and processed using SlideBook software version 4.0.1.16 [EC] (Intelligent Imaging Innovations, Denver, CO).
NF-
PMNs (20 x 106) were resuspended in 400 µL sonication buffer (20 mM imidazole, pH 7.4; 0.25 M sucrose; 5 mM EGTA; 2.5 mM MgCl2) supplemented with protease and phosphatase inhibitors, sonicated on ice (Fisher Scientific [Hampton, NH] Sonic Dismembrator Model 100, setting 2, 20 x 2-second pulses), salt-extracted (400 mM NaCl, 20 minutes, 4°C), and centrifuged (18 380g, 15 minutes, 4°C). Sonicate (15 µg) was assayed by TransAM p65 kit (Active Motif) per the manufacturer's instructions. Cytoplasmic fractions were normalized by protein assay25 and immunoblotted with rabbit anti-I Rho GTPase pulldown PMNs (20 x 106) were lysed in 500 µL (50 mM Tris pH 7.5, 10% glycerol, 1% Ipegal in 0.9% NaCl), clarified by centrifugation, precleared with GST-Sepharose (15 µg, 1 hour, 4°C), and then incubated for 2 hours with glutathione-Sepharose beads and 15 µg either GST or GST fusion protein (wt RhoA, L63RhoA). Beads were then washed 3 times, eluted in 1 x Laemmli buffer, and boiled. Statistical analysis Data are reported as mean ± SE. In analyses involving multiple comparisons (Figures 1A-B; 2A,C,E; 3D,G), ANOVA was used (2-way ANOVA with Bonferroni post test analysis, or stratified 1-way ANOVA with Tukey post test analysis). In other cases, analysis was performed using a 2-tailed Student t test (Prism; GraphPad Software, San Diego, CA). P less than .05 was considered statistically significant.
LPS activates RhoA and ROCK by a p38 MAPK-mediated pathway
Our group has previously reported that p38 is the only MAPK activated by LPS in the suspended human PMN and that p38 regulates LPS-induced NF- To address this issue, we tested for LPS-induced activation of RhoA in human PMN lysates, using a Rhotekin-Rho Binding Domain (RBD)–GST-Sepharose pulldown.26 Active RhoA was detected in the resting PMN, and LPS induced further accumulation of GTP-RhoA in a time-dependent fashion, first noted by 15 to 30 minutes (Figure 1A). Activation of RhoA was p38 dependent, as revealed by the use of SB203580, a p38 inhibitor. We have previously reported p38-dependent activation of the Rho GTPase Cdc42 in the human PMN within 10 to 15 minutes of LPS exposure.30 As this is earlier than RhoA activation (Figure 1A), and Cdc42 has been reported to be upstream of RhoA,31 we next questioned whether Cdc42 lies upstream of RhoA in the PMN's LPS signaling cascade. No cell-permeant inhibitors specific for Cdc42 are available. Hence, we used BioPORTER reagent to protein-transduce the Cdc42 binding domain of the Cdc42 effector, Wiskott Aldrich syndrome protein (WASP-CRIB), a method previously reported specifically to inhibit Cdc42 in the human PMN by sequestering GTP-Cdc42.24 As a negative control, we transduced a mutant, non–Cdc42-binding WASP-CRIB. As shown in Figure 1B, LPS-induced RhoA activation was inhibited in PMNs pretreated with wild-type but not mutant WASP-CRIB protein, confirming that Cdc42 lies upstream of RhoA in the LPS signaling pathway. As we have previously reported basal activation of Cdc42 in the resting human PMN,30 the lack of any effect of WASP-CRIB on GTP-RhoA expression in the resting PMN (Figure 1B) suggests that Cdc42-independent mechanisms are responsible for basal RhoA activation. In support of this, transduced constitutively active L61Cdc42 was not sufficient to activate RhoA in the resting PMN (Figure 1C).
RhoA, by contrast, inhibited basal activation of Cdc42 in the resting PMN. In the resting cell, C3 transferase, a specific Rho inhibitor that ADP-ribosylates RhoA on Asn41, leading to its gel-retardation and degradation32 (Figure 1D) and thereby modifies the actin cytoskeleton (Figure 1D), was sufficient to induce modest Cdc42 activation (Figure 1D), as assessed by a p21-binding domain pulldown.27 By contrast, C3 had no effect on either basal or LPS-induced p38 activation (Figure 1D). Taken together with the preceding findings, this suggests that (1) Cdc42 mediates LPS-induced, but not spontaneous activation of RhoA; (2) Cdc42 is not sufficient for RhoA activation in the resting PMN; and (3) RhoA suppresses Cdc42 in the resting PMN.
Finally, to confirm further the downstream significance of RhoA activation, we tested for activation of a prototypical Rho-specific effector, ROCK
In summary, these data suggest contrasting suppressive versus stimulatory roles for RhoA in the resting and LPS-exposed human PMN: (1) inhibition of Cdc42 by RhoA in the resting state and (2) a pathway leading from p38 to Cdc42 to RhoA to ROCK LPS-induced p38 activation and Rho GTPases In contrast to our findings, existing reports of Rho GTPases in other cell types place these molecular switches upstream of MAP kinases.35,36 Nevertheless, neither of 2 surrogate assays of p38 activation, phosphospecific immunoblotting and in vitro kinase assay, revealed diminution of p38 activation in the presence of a pan-Rho GTPase inhibitor, C difficile toxin B16 (Figure 1F), at the inhibitor concentration tested. Similar results were noted with C3 transferase (Figure 1D; data not shown). Alternatively, RhoA has also been reported to be functionally upstream of ERK by regulating its nuclear/cytoplasmic translocation,37 an effect that might conceivably be important to transcriptional and translational regulation. Immunoblotting revealed no effect of either toxin B or Y27632, a ROCK inhibitor,38 on nuclear localization of either PO4-p38 or total p38 (data not shown). Thus, in contrast to other cell types, Rho GTPases and ROCK, as tested by the inhibitors toxin B and Y27632 respectively, do not appear to regulate either the activity or nuclear/cytoplasmic localization of p38 in the LPS-stimulated human PMN.
Cdc42 regulates TNF
We have previously reported that p38 MAPK regulates LPS-induced TNF
Having identified a regulatory role for Cdc42 in TNF transcript expression in the resting PMN, we next sought to characterize further the underlying mechanism. TNF transcription is strongly NF-B dependent.41 We are unaware of any reports associating Cdc42 with NF-B activation in the PMN. To address this, we assayed the effect of L61Cdc42 on binding of the p65 subunit of NF-B to its cognate DNA sequence. Using a sandwich ELISA of p65 binding to immobilized B oligonucleotide sequence (p65 TransAM; Active Motif), we observed that L61Cdc42 was sufficient to induce p65 activation in the PMN (Figure 2E). Because proteasomal degradation of cytoplasmic IB is a critical mechanistic step in canonical NF-B activation,42 we also evaluated IB expression in cytoplasmic fractions of L61Cdc42-transduced PMNs. L61Cdc42 but not GST control induced IB degradation (Figure 2F). These results suggest that Cdc42 is sufficient to activate NF-B in the resting human PMN, and, moreover, that Cdc42 may induce TNF gene expression through a transcriptional mechanism involving NF-B. Consistent with our results showing no effect of WASP-CRIB on LPS-induced TNF gene expression, no consistent effect of WASP-CRIB was seen on LPS-induced IB degradation (data not shown).
RhoA suppresses TNF
We next sought to define the independent role of RhoA in regulation of TNF
Inhibition of RhoA has been described to disrupt the actin cytoskeleton (Figure 1D)47,48 and cytoskeletal remodeling to modulate signal transduction.49 Moreover, cytochalasins, which are actin depolymerizing agents, have long been known to "prime" leukocytes for certain proinflammatory functions, such as release of O  .50,51 Hence, we queried whether the observed induction of TNF by RhoA inhibition in the resting cell might be mediated by cytoskeletal disruption. Treatment of the resting PMN with cytochalasin B (Figure 3E) or cytochalasin D (data not shown) did not induce TNF transcript or protein (data not shown) expression. These findings suggest that the TNF-inducing effects of C3 and Rhotekin-RBD are not consequences of actin remodeling. As Cdc42 has also been tightly linked to actin polymerization in the human PMN,52,53 the absence of TNF induction with WASP-CRIB (Figure 2A-B) corroborates this conclusion.
Because C3 transferase activated Cdc42, and both C3 and constitutively active Cdc42 induced TNF
RhoA suppresses NF-
NF- We next queried whether RhoA might physically associate with p65, and whether such an association might be sensitive to RhoA activation state. To address this question, we exposed PMNs to a time course of LPS and then performed pulldowns on lysates, using Sepharose-conjugated GST fusions of wt RhoA and constitutively active L63RhoA. As depicted in Figure 4A, p65 coprecipitated with wt RhoA, but minimal coprecipitation was observed with L63RhoA.
ROCK, a RhoA effector, positively regulates LPS-induced TNF expression
The marked activation of NF-
The prototypical acute-response cytokine TNF is produced by multiple different cell types and plays a variety of roles, beneficial and deleterious, in inflammation and host defense.1,2 Perhaps as a consequence, TNF induction is precisely controlled,3–5 with important differences among cell types.5,7,8,56 Fundamental differences exist between RhoA signaling in the human PMN and transfectable cell lines, such as HEK293s.57 Hence, these lines cannot be used to model RhoA signaling in the human PMN. Moreover, the human PMN differs in several important ways from other leukocytes, likely because of its unique role as a short-lived, acute-response effector cell.15,21 An improved understanding of the specific regulatory mechanisms underlying TNF induction in the PMN provides an opportunity for better defining the controls underlying the "resting" and "activated" states of the PMN itself.
Rho GTPases have been extensively studied in the PMN, wherein they have been classically described to mediate cytoskeletal-related proinflammatory functions.17 In the present study, we applied protein transduction tools to the human PMN to describe novel roles for RhoA and Cdc42 in TNF
We propose a working model (Figure 5) to synthesize our present findings in the context of previous literature. In the resting cell, inhibition of p38 and Cdc42 do not recapitulate the positive effects on TNF expression observed with inhibition of RhoA, likely because RhoA activity in the resting cell is p38- and Cdc42-independent (Figures 1A-B and 5). This differs from the LPS-stimulated state, wherein RhoA activity is both p38 and Cdc42 dependent (Figure 1A-B). These findings suggest that a condition (eg, an activated molecule) characteristic of the LPS-stimulated, but not resting PMN, may be required for p38 and Cdc42 to regulate (ie, be "upstream" of) RhoA activity. In the context of LPS stimulation, inhibition of p38 and Cdc42 both reduce TNF protein8 (Figure 2), possibly through regulation of ROCK downstream (Figure 5). Although the predominant regulatory effect of p38 on TNF may be through this Cdc42-mediated pathway, our previous finding of subtle, transient effects of p38 on LPS-induced TNF transcripts and on NF-B activation8 (neither of which were observed with Cdc42 inhibition in the present study) suggests that p38 may also regulate TNF through Cdc42- and RhoA-independent means (Figure 5), for example, through MK2, as has been reported.59–61 Nevertheless, our data do not exclude a role for Rho GTPases in LPS-induced NF-B activation in the human PMN.
In vivo, the human PMN is unlikely to encounter LPS as a solitary stimulus in isolation from cytokines or other pathogen-associated molecular patterns. We speculate that the multiple-level, counter-regulatory controls that we have observed may allow for integration of extracellular signals at hierarchical signaling hubs that permit for contextual control over TNF Limitations of the present study should be noted. We cannot be certain that transduced L61Cdc42 has all of the necessary features of endogenous Cdc42 for RhoA activation or that we sampled the correct time point for RhoA activation following L61Cdc42 transduction. Because we did not confirm toxin B stoichiometry or assay all aspects of p38 activation, we cannot be fully certain that p38 activation is Rho independent in all respects. How closely the ex vivo isolated PMN models the in vivo resting PMN was not addressed. The RhoA effector(s) operative in the ROCK-independent suppressive mechanism in the resting PMN was not identified in the present study and will be the focus of future efforts. Of interest, examples exist in the literature of both cooperative and antagonistic interactions among RhoA effectors,18,19,62 of coregulatory proteins such as CNK1 that direct signaling specificity downstream of RhoA,63 and of proteins that inhibit specific RhoA effectors.64 Hence, the potential exists for sophisticated cellular control over signaling specificity downstream of RhoA. Moreover, the existence of multiple independent RhoA effectors provides precedence for distinct, and even contrary, roles for RhoA under different states of cellular activation, as seen in the present study.
Finally, our data may suggest an additional mechanism of clostridial pathogenesis: "inappropriate" elaboration of proinflammatory cytokines. Nevertheless, the potential for dynamic RhoA inhibition in biologic systems exists not only through the action of bacterial toxins but also through host endogenous proteins. For example, cadherin engagement,65 serine phosphorylation,66 tenascin-C,67 and c-AMP–dependent protein kinase68 are reported to inhibit RhoA and the Rnd proteins to inhibit RhoA and ROCK.69,70 Such reports raise the intriguing possibility that TNF
Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Michael B. Fessler, National Institute of Environmental Health Sciences, 111 T. W. Alexander Dr., PO Box 12233, MD D2-01, Research Triangle Park, NC 27709; e-mail: fesslerm{at}niehs.nih.gov.
We thank Klaus Hahn for providing the WASP-CRIB constructs, and Yu Hong Liu for assistance with recombinant protein expression. This work was supported by the American Heart Association (grant 0275035N) (M.B.F.) and the National Institutes of Health (grant 5R01HL061407-08) (G.S.W.), (grant 5P01HL68743-04) (J.A.N.), and (grant HL67179) (P.G.A.).
Submitted March 27, 2006; accepted September 8, 2006.
Prepublished online as Blood First Edition Paper, October 3, 2006
DOI: 10.1182/blood-2006-03-012898
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Abstract
Introduction
