ARTHRITIS & RHEUMATISMVol. 52, No. 4, April 2005, pp 1297–1304DOI 10.1002/art.20984© 2005, American College of Rheumatology

Innate Immunity and Arthritis

Neutrophil Rac and Toll-Like Receptor 4 Expression Define Outcomes inInfection-Triggered Arthritis

Xiang Zhang,1 Michael Glogauer,2 Fei Zhu,2 Tae-Hwan Kim,3

Basil Chiu,1 and Robert D. Inman1

Objective. The role of innate immunity inChlamydia-induced arthritis has not been defined. Thepurpose of this study was to examine the role ofneutrophils in experimental arthritis in mice with tar-geted elimination of the small GTPases Rac1 and Rac2,as well as the role of Toll-like receptors (TLRs) in thismodel.

Methods. Arthritis was induced by intraarticularinoculation of synoviocyte-packaged Chlamydia tracho-matis. The degree of arthritis was assessed according tojoint swelling and pathology scores. The persistence ofChlamydia in joints was assessed by immunoassay. Theexpression of TLR-2 and TLR-4 in neutrophils wasdetected by semiquantitative reverse transcription–polymerase chain reaction.

Results. In the acute phase, wild-type mice devel-oped more severe arthritis than did Rac-deficient mice,with abundant infiltration of neutrophils into the joint.In the chronic phase, the Rac-deficient mice developedmore severe arthritis and demonstrated defective clear-ance of the pathogen from the joint. In vitro stimulationof neutrophils with Chlamydia up-regulated the expres-

sion of TLR-4, but not TLR-2, in wild-type mice. How-ever, neutrophils from Rac-deficient mice did not showthis up-regulation of TLR-4. Sustained TLR-4 expres-sion in neutrophils was found to be dependent on theexpression of Rac. Mice genetically deficient in TLR-4demonstrated more severe arthritis than did the con-trols. Thus, Rac expression plays a profound role ininfection-triggered arthritis and demonstrates a bi-modal influence on the disease process, exacerbatingacute joint inflammation but controlling chronic arthri-tis. Rac deficiency was associated with diminishedTLR-4 expression, impaired host clearance of the patho-gen, and more severe chronic arthritis.

Conclusion. In infection-triggered arthritis, in-nate immunity plays a critical role. Effective host clear-ance of an arthritogenic pathogen depends on intactexpression of Rac and appropriate expression of TLR-4by neutrophils.

The triggering of joint inflammation by infectiousagents has been well established in the case of reactivearthritis (1,2). Infection with the obligate intracellularpathogen Chlamydia trachomatis is a common anteced-ent for reactive arthritis (3). We previously establishedan animal model of Chlamydia-induced arthritis thatclosely mirrors clinical reactive arthritis (4). However,the role of neutrophils in such infection-triggered arthri-tis remains undefined. Since neutrophils are central toinnate immunity and represent the host’s first line ofdefense, their role in the initial phase of this process mayaffect the outcome of the joint inflammation in the laterphase.

The small GTPases Rac1 and Rac2 are expresseddifferentially in cells: Rac1 is ubiquitously expressed inall cells while Rac2 is expressed only in cells of thehematopoietic lineage, including neutrophils. Although

Dr. Zhang’s work was supported by the Arthritis Center ofExcellence and the Canadian Institutes of Health Research. Drs.Glogauer and Inman’s work was supported by the Canadian Institutesof Health Research.

1Xiang Zhang, MD, PhD, Basil Chiu, PhD, Robert D. Inman,MD: Toronto Western Hospital, and University of Toronto, Toronto,Ontario, Canada; 2Michael Glogauer, DDS, Fei Zhu, MSc: Universityof Toronto, Toronto, Ontario, Canada; 3Tae-Hwan Kim, MD, PhD:Toronto Western Hospital, and University of Toronto, Toronto,Ontario, Canada, and Hospital for Rheumatic Diseases, HanyangUniversity, Seoul, South Korea.

Address correspondence and reprint requests to Robert D.Inman, MD, Division of Rheumatology, Toronto Western Hospital,ECW8-005, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada.E-mail: Robert.inman@uhn.on.ca.

Submitted for publication July 5, 2004; accepted in revisedform January 11, 2005.

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human neutrophils mainly express Rac2, comparableamounts of Rac1 and Rac2 are expressed on murineneutrophils (5). Recent studies have shown that Rac1and Rac2 are both important regulators of actin-mediated neutrophil functions, including recruitment ofinflammatory cells, migration to chemotactic stimuli,and chemoattractant-mediated actin assembly (6–8).Moreover, Rac2 is specifically required for the genera-tion of NADPH oxidase in neutrophils (7,8).

In the present study, we addressed the role ofneutrophils in Chlamydia-induced arthritis using micewith targeted elimination of Rac1 and Rac2 (Rac–/–).We also sought to determine whether the expressionpattern of neutrophil Toll-like receptors (TLRs) in thepresence or absence of Rac proteins after challenge withChlamydia is involved in the development of thispathogen-triggered joint inflammation.

MATERIALS AND METHODS

Mice. We applied a gene-targeting approach that se-lects for the specificity of the Lys Cre promoter of neutrophilsand monocytes (9), yielding mice in which Rac1 was selectivelydeleted from neutrophils and monocytes (7,10). Rac1–/– mice(Rac1c/–LysMcre) were bred with Rac2–/– mice (6). The result-ing offspring were bred for at least 6 generations to generateoptimal breeding pairs (Rac1c/-LysMcreRac2�/– � Rac1c/–

LysM–Rac2�/–) that would enable the generation of Rac1/Rac2 double-deficient (Rac1c/–LysMcreRac2–/–) and wild-type(Rac1�/�LysMcreRac2�/�) mice from the same litters. For thesake of simplicity, we have used “Rac-deficient” in the text and“Rac–/–” in the figures to represent Rac1/Rac2 double-deficient mice. This breeding strategy allows for the control ofbackground variations. In a recent study (10), we confirmedthe absence of Rac1 and Rac2 protein expression in therespective knockout mice. C57BL/10ScN (having completedeletion of the tlr4 gene) and C57BL/10J (the respectivewild-type control), were purchased from Jackson laboratory(Bar Harbor, ME).

All experiments were performed with 8–12-week-oldmice of both sexes. At each time point, at least 6 mice in eachgroup were killed. All procedures were carried out in accor-dance with the Guide for the Humane Use and Care ofLaboratory Animals and were approved by the University ofToronto Animal Care Committee and the University HealthNetwork Animal Care Committee.

Induction of arthritis. The culture and inoculation ofChlamydia trachomatis serotype L2 was conducted as previ-ously described in a rat model (4). Arthritis was induced by asingle intraarticular injection of synoviocyte-packaged C tra-chomatis. Synoviocytes for this study were generated fromLewis rat knee joints as previously described (4). We used ratsynovial fibroblasts for our studies for the following reasons.Pilot studies indicated that the Chlamydia grew much moreproductively in the rat synovial fibroblasts than in mouse Lcells. To exclude possible cross-species confounding variables,

we carried out preliminary studies using uninfected rat syno-viocytes for control injections. Injection of the mouse joint withthese cells induced no inflammation clinically or histopatho-logically and the profile was indistinguishable from that foundafter an intraarticular injection of sterile saline.

For reproducible and accurate injection of the mousejoint, a 30-gauge needle connected to a microinjection pump(CMA/100 Microinjection Pump; Carnegie Medicin, Stock-holm, Sweden) was used. We injected 20 �l of the preparation(104 cells containing 2 � 104 chlamydial inclusion-formingunits) into mouse knee joints. The mice were killed on days 1,2, and 14 postinjection. The degree of arthritis was assessedaccording to the degree of joint swelling and histopathologicchanges in the synovium.

Histopathology and immunohistochemistry. For his-topathologic assessment, the joints were fixed in bufferedformalin phosphate, decalcified, and stained with hematoxylinand eosin. Each joint was assigned a histopathologic scoreaccording to the Yang-Hamilton grading system, which as-sesses inflammatory cell infiltrates, joint space exudates, syno-vial hyperplasia, pannus formation, and cartilage/bone damage(11,12). We scored 2 sections from each joint. The statisticalanalysis was conducted on a group of 6 animals, each of whichhad 2 joints analyzed.

Immunohistochemistry was used to detect neutrophilsin the joints, using a biotinylated monoclonal antibody specificfor murine neutrophils (CL8993B; Cedarlane, Hornby, On-tario, Canada). Briefly, formalin-fixed, paraffin-embeddedjoint tissues were cut at thickness of 4 �m and placed onpositively charged slides. The slides were dried overnight at37°C, then deparaffinized and rehydrated through xylenes andgraded ethanols to water. All slides were blocked for endoge-nous peroxidase for 15 minutes in a 3% hydrogen peroxidesolution in distilled water. The sections were then washed andplaced in Tris buffer. The sections were incubated for 1 hour atroom temperature with the biotinylated primary antibodydiluted at 1:40. Sections were rinsed in Tris buffer and thenincubated for 30 minutes in Vectastain Elite (Vector, Burling-ton, Ontario, Canada) at a 1:200 dilution. The peroxidasereaction was visualized with diaminobenzidine tetrahydrochlo-ride. The sections were then counterstained with hematoxylin,dehydrated, cleared, and mounted in Permount.

Clearance of Chlamydia from body tissues. The persis-tence of the injected Chlamydia in the joints and spleens ofmice was assessed with the immunoassay kit IDEIA PCEChlamydia from DakoCytomation (Cambridge, UK). After theanimals were killed, the spleens were collected, homogenized,filtered to remove any tissue debris, and the cells werecollected by centrifugation. Synovial tissues were dissectedfrom the knee joint. The cell pellets and the joint tissues wereprocessed for detection of Chlamydia according to the manu-facturer’s guidelines. All procedures were conducted understrict sterile conditions.

Preparation of neutrophils. Murine neutrophils wereisolated from bone marrow as previously described (7). Briefly,femurs and tibias were removed and separated from muscles.Both ends of the bone were cut, and the bone marrow cellswere flushed into the culture media. The cells were layeredonto a discontinuous Percoll gradient of 82%/65%/55%. Ma-

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ture neutrophils were collected at the 82%/65% interface, with�85% cells being neutrophils, as confirmed by Wright-Giemsastaining, and �90% cells being viable, as determined by trypanblue staining. These neutrophils were stimulated with C tra-chomatis elementary bodies (Microbix Biosystems, Toronto,Ontario, Canada) at a neutrophil to Chlamydia ratio of 1:10 for16 hours. Expression of TLR-2 and TLR-4 on these cells wasanalyzed quantitatively. The absence of Rac expression byneutrophils from Rac-deficient mice was confirmed by immuno-blot analysis (data not shown).

Semiquantitative reverse transcription–polymerasechain reaction (RT-PCR). Total RNA was isolated fromneutrophils (with or without chlamydial stimulation) by usingTRIzol reagent (Invitrogen, Burlington, Ontario, Canada).Samples of total RNA (1 �g) were reverse transcribed into thefirst-strand complementary DNA (cDNA) using 1.0 �l ofoligo(dT) (0.5 �g/�l), 2.0 �l of 10 mM dNTPs, 1.0 �l of 50 mM

MgCl2, 1.0 �l of SuperScript II RNase H– reverse transcriptase(200 units/�l), 0.5 �l of RNase-Out recombinant ribonucleaseinhibitor (40 units/�l; Invitrogen), 2.0 �l of 10� amplificationbuffer, and double-distilled H2O to bring the total volume to20 �l. The mixture was incubated at 37°C for 30 minutes. PCRwas performed with specific primers specific for mouse TLR-2,TLR-4, and L32. The PCR reaction started with 4 �l of reversetranscription mixture (cDNA), 1.5 �l of 10� PCR buffer, 25pmoles of primers, and ddH2O to a total volume of 17.5 �l,with incubation at 95°C for 1 minute. The temperature wasmaintained at 80°C in order to add 1.0 �l of 1.5 units of TaqDNA polymerase (5 units/�l; Invitrogen). The PCR reactionwas continued for 22 cycles (94°C for 15 seconds, 55°C for 15seconds, 72°C for 25 seconds, and a final elongation step of 7minutes at 72°C) for L32, 30 cycles for TLR-2, and 30 cycles forTLR-4 with annealing temperature of 57°C.

Primers used were as follows: for L32, 5�-CATGGC-TGCCCTTCGGCCTC-3� (upstream) and 5�-CATTCTC-TTCGCTGCGTAGCC-3� (downstream); for TLR-2, 5�-CAGCTTAAAGGGCGGGTCAGAG-3� (upstream) and 5�-TGGAGACGCCAGCTCTGGCTCA-3� (downstream); and forTLR-4, 5�-AGTGGGTCAAGGAACAGAAGCA-3� (upstream)and 5�-CTTTACCAGCTCATTCTCACC-3� (downstream).

Figure 1. Development of arthritis after intraarticular injection ofChlamydia trachomatis in wild-type and Rac–/– mice (n � 6 per groupper time point). For joint swelling measurements, each joint was fixedin buffered formalin phosphate, and the overlying skin was removedbefore measuring. Joint swelling (in mm) was measured with calipers.The histopathologic score for each knee joint was assigned accordingto the Yang-Hamilton 5-criteria grading system, based on the presenceof inflammatory cell infiltrate, joint space exudate, synovial hyperpla-sia, pannus formation, and cartilage/bone damage. Values are themean and SEM. � � P � 0.05; ��� � P � 0.001.

Figure 2. Immunohistochemical staining of neutrophils in synovialtissues from the joints of wild-type and Rac–/– mice 24 hours afterinjection of Chlamydia trachomatis. Abundant neutrophils werepresent (brown staining) in the joints of wild-type mice, whereasneutrophil infiltration was diminished in Rac–/– mice (original magni-fication � 150).

INNATE IMMUNITY IN INFECTION-TRIGGERED ARTHRITIS 1299

Statistical analysis. Results of joint swelling, his-topathologic scoring, and PCR analyses were expressed as themean � SEM. To determine the statistical significance, Stu-dent’s t-test was performed.

RESULTS

Rac expression and exacerbation of the severityof arthritis in the acute phase. In the acute stage ofChlamydia-induced arthritis, 24–48 hours after injectionof the joint, wild-type mice developed more severearthritis than did Rac-deficient mice (Figure 1). Themost significant differences were observed at 24 hourspostinjection, when wild-type mice had more markedswelling (3.6 mm versus 3.0 mm in Rac–/– mice; P �0.001) and higher histopathologic scores (5.2 versus 2.8in Rac–/– mice; P � 0.05) compared with Rac-deficient

mice. Immunohistochemistry showed abundant neutro-phils infiltrating the synovial tissue in wild-type mice,whereas significantly fewer neutrophils were present inall of the Rac-deficient mice (Figure 2). No neutrophilswere seen in the control noninjected joints (data notshown).

These results indicated that in Rac-deficientmice, the neutrophils were impaired in their migrationinto the joint, a finding consistent with the notion thatRac proteins are important in the regulation of neutro-phil chemotaxis. It was evident that this impaired neu-trophil influx into the joint in the Rac-deficient micewas associated with less severe arthritis in the acutephase. Our results demonstrated that in the acute stage,the severity of Chlamydia-induced arthritis correlatedwith the degree of neutrophil infiltration in the jointsand that the Rac proteins were the critical controlelements of this aspect of the innate immune response ofthe host.

Rac expression and mitigation of the severity ofarthritis in the chronic phase. In the chronic phase, thearthritis was most severe in Rac-deficient mice (jointswelling 3.9 mm; histopathologic score 8.8) comparedwith wild-type mice (joint swelling 3.3 mm; histopatho-logic score 4.5) (Figures 1 and 3). In terms of cellmorphology, hematoxylin and eosin stained sectionsshowed that the infiltrating cells on day 14 consistedpredominantly (�65%) of neutrophils, with mono-nuclear cells in the minority (�35%). It seemed that thecontinuous influx of neutrophils for 14 days resulted inmassive cartilage damage (Figure 3). Thus, in contrast to

Figure 3. Histopathologic changes in the joints of wild-type andRac–/– mice 14 days after injection of Chlamydia trachomatis. Thesynovitis was most severe in Rac–/– mice, whereas the severity ofarthritis was diminished in wild-type mice (original magnification �200).

Figure 4. Detection of chlamydial antigen in the joints of wild-typeand Rac–/– mice after intraarticular injection of Chlamydia trachomatis.Values are the percentages of mice testing positive for the presence ofChlamydia in synovial tissues. A total of 20 joints were examined in thisexperiment (n � 4–6 mice per group).

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the acute phase, the findings indicate that at this timepoint, Rac expression plays a protective role in thehost–pathogen interaction with respect to joint injury.These results suggest that deletion of Rac protein im-poses a functional defect that might impair effective host

clearance of Chlamydia, with accelerated joint inflam-mation in the chronic phase as a result.

Dominant role of Rac in host clearance of thepathogen. As detected in joint tissues by immunoassay,Chlamydia were recovered in 50% of Rac-deficient miceand in 25% of wild-type mice at 24 hours postinjection(Figure 4). However, after 48 hours, wild-type micecleared Chlamydia more efficiently, and fewer joints(20%) were positive for Chlamydia. In contrast, increas-ing numbers of Chlamydia-positive joints were seen inRac-deficient mice. These results suggested that Racexpression plays an important role in host elimination ofthe pathogen. This may relate to the recent demonstra-tion that Rac2 is specifically required for activation ofthe neutrophil NADPH oxidase (7,8). At 14 days postin-jection, all joints were negative for persisting chlamydialantigen. No chlamydial antigen was detected in anyspleen samples (data not shown).

Figure 5. Expression of Toll-like receptor 2 (TLR-2) and TLR-4 inneutrophils obtained from wild-type and Rac–/– mice. Neutrophilswere stimulated in vitro for 16 hours with chlamydial elementarybodies. A, Total RNA was extracted from parallel wells at 16 hours,and reverse transcription–polymerase chain reaction (PCR) was per-formed by using primers specific for TLR-2, TLR-4, and L32. Molec-ular markers are shown in the first lane. B, PCR products for TLR-2and TLR-4 were normalized to the L32 PCR product. Values are themean and SEM. �� � P � 0.01; ��� � P � 0.001.

Figure 6. Development of arthritis in Toll-like receptor 4 (TLR-4)–deficient mice. Joints were harvested on day 5 after injection ofChlamydia trachomatis. Joint swelling (in mm) was measured withcalipers. There were significant differences in joint swelling andhistopathology scores between TLR-4–deficient C57BL/10ScN miceand wild-type C57BL/10J mice. Values are the mean and SEM.� � P � 0.05.

INNATE IMMUNITY IN INFECTION-TRIGGERED ARTHRITIS 1301

Dependence on Rac expression for sustainedTLR-4 expression in neutrophils. TLR-4 was constitu-tively expressed on resting neutrophils in both wild-typeand Rac-deficient neutrophils. TLR-4 expression wasstrongly up-regulated in wild-type neutrophils after 16hours of chlamydial stimulation (Figures 5A and B).However, this up-regulation was significantly attenuatedin Rac-deficient neutrophils, indicating that Rac playsan essential role in Chlamydia-induced up-regulation ofTLR-4. These results also suggested that the TLR-4expression upon chlamydial stimulation might relatedirectly to the host clearance of the pathogen from thejoint and the resulting severity of chronic joint inflam-mation in the chronic phase. TLR-2 was also expressedon resting neutrophils (Figures 5A and B). TLR-2expression was up-regulated upon chlamydial stimula-tion in both wild-type and Rac-deficient neutrophils, butno differences were seen between the 2 groups (Figures5A and B), indicating that TLR-2 up-regulation isindependent of Rac.

Deficiency of TLR-4 expression resulting in moresevere arthritis. Since the results above pointed toTLR-4 as being critical in the pathogenesis ofChlamydia-induced arthritis, we examined this model ina strain of TLR-4–deficient mice, C57BL/10ScN, whichhave complete deletion of the tlr4 gene. Our resultsindicated that C57BL/10ScN mice developed signifi-cantly more severe arthritis than the respective controlmice, C57BL/10J, as evidenced by more joint swelling(3.52 � 0.11 mm versus 3.20 � 0.04 mm; P � 0.05) andhigher histopathologic scores (8.8 � 0.67 versus 6.5 �0.26; P � 0.05) (Figure 6). These results suggested thatdefects in TLR-4 expression result in impaired clearanceof the pathogen and enhanced joint inflammation.

DISCUSSION

The role of neutrophils in infection-triggeredarthritis is not well defined, but experimental modelshave emphasized the critical role that these cells can playin joint inflammation. Our study may have some paral-lels with streptococcal cell wall–induced arthritis inwhich depletion of neutrophils reduced joint inflamma-tion in the acute phase (13). Moreover, similar conclu-sions have been drawn with regard to the murine K/BxNarthritis model, in which transfer of serum from K/BxNmice into recipient mice induces a rapid inflammatoryresponse in the joint. This, too, is dependent on neutro-phils in the acute phase, since the absence of neutrophils

in recipient mice conferred resistance to the inflamma-tory effects of K/BxN serum (14).

The expression and functional regulation ofTLRs on human neutrophils have been examined in arecent study (15). In our study, both TLR-2 and TLR-4were expressed on resting murine neutrophils. It is wellknown that TLR-2 recognizes the ligand of gram-positive bacteria, whereas TLR-4 recognizes gram-negative bacteria (16,17). It might therefore be expectedthat Chlamydia would favor TLR-4 interaction, sinceChlamydia is a gram-negative intracellular pathogen.However, it has been shown that the activation ofmurine dendritic cells upon contact with Chlamydiapneumoniae is largely dependent on TLR-2 and is de-pendent only to a minor extent on TLR-4 (18). Incontrast, it has been reported that the activation ofmurine macrophages and human endothelial cells bychlamydial heat-shock protein 60 is dependent onTLR-4 (19). In our study, the antimicrobial function ofmurine neutrophils upon stimulation with C trachomatiswas largely dependent on TLR-4, since the clearance ofC trachomatis was attenuated in the setting of TLR-4deficiency. This notion is supported by the recent clinicalobservation that impaired TLR-4 expression (induced byanti–tumor necrosis factor therapy) confers increasedsusceptibility to intracellular bacterial infection (20).Moreover, this is the first study to show that sustainedTLR-4 expression in neutrophils upon contact with Ctrachomatis is primarily dependent on Rac, since TLR-4up-regulation was abrogated in neutrophils deficient inRac protein. It is also noteworthy that TLR-4 is theprincipal regulator of neutrophil survival (21).

The role of TLRs in joint destruction is currentlyunder investigation. A recent study using the K/BxNmodel of arthritis suggested that TLR-4 exacerbates theseverity of arthritis in that system (22). In contrast, ourstudy represents the first evidence that TLR-4 can playan important protective role in the course of infection-triggered arthritis.

Our results demonstrated that Rac expressionplays a profound dual role in Chlamydia-triggered ar-thritis. In the acute phase, Rac functions to exacerbatejoint inflammation, since delayed neutrophil migrationinto the joints resulted in less joint inflammation. In thechronic phase however, Rac serves to alleviate arthritis,since Rac-deficiency resulted in more severe arthritis.This is probably due to the defective host clearance ofChlamydia, as shown in Figure 4. We attempted toaddress the microbial load on day 14, but the resultsappeared to be below the limits of detection of the

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antigen assay we used. But despite the failure to detectpersisting pathogen, the inflammation persisted at thistime, suggesting that what began as an infectious triggercould become a culture-negative chronic arthritis withtime. This was our experience with the rat model in thissystem (4). Previous studies have demonstrated a lack ofbactericidal NADPH oxidase in neutrophils from Rac-deficient mice, suggesting that this might contribute tothe defective host clearance of Chlamydia that we ob-served (7,8). In our study, we found that Rac deficiencywas also associated with defective expression of TLR-4(Figure 5). Diminished TLR-4 expression may result in afunctionally defective innate immune system that leadsto the persistence of bacteria in the joints and thesubsequent development of more severe arthritis, asseen in the TLR-4–deficient mice (Figure 6).

The mechanism whereby Rac may augmentTLR-4 expression in neutrophils is not currently known,and we recognize the limitations of using an in vitrosystem as we have done. However, the decreased expres-sion of TLR-4 was confirmed by molecular techniques.It is widely recognized that there are no anti-murineTLR-4 antibodies suitable for use in blotting studies, andso, we were not able to demonstrate the expression ofTLR-4 protein in a Western blotting experiment usingcommercially available anti–TLR-4 antibodies. Al-though fluorescence-activated cell sorter analysis of mu-rine macrophages for TLR-MD-2 expression has beenreported by previous investigators (23), comparableanalysis of murine neutrophils has not been demon-strated to date. It has also been reported that TLR-2signals NF-�B via a Rac1-regulated pathway (24). Fur-ther studies into the interaction between TLR-4 and itsdownstream Rac-regulated pathway may reveal impor-tant insights into neutrophil antimicrobial function fol-lowing bacterial invasion. A better understanding ofthese fundamental mechanisms may also suggest newtherapeutic interventions for inflammatory joint disease.

ACKNOWLEDGMENTS

We thank Dr. Kenneth Pritzker, Maria G. Mendes,Patricia Wegrynowski, and the Department of Pathology,Mount Sinai Hospital, for technical help with the histology andimmunohistochemistry. We also thank Drs. Chi P. Wu, LiangZhang, and Yongqiang Wang for technical assistance.

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