ARTHRITIS & RHEUMATISMVol. 21, No. 7, July 2005, pp 2109–2119DOI 10.1002/art.21129© 2005, American College of Rheumatology

Dysregulation of Chemokine Receptor Expression and Functionby B Cells of Patients With Primary Sjogren’s Syndrome

Arne Hansen,1 Karin Reiter,1 Till Ziprian,1 Annett Jacobi,1 Andreas Hoffmann,1

Mirko Gosemann,1 Jurgen Scholze,1 Peter E. Lipsky,2 and Thomas Dorner1

Objective. To assess whether abnormal chemo-kine receptor expression and/or abnormal responsive-ness to the cognate ligands might underlie some of thedisturbances in B cell homeostasis characteristic ofprimary Sjogren’s syndrome (SS).

Methods. Chemokine receptor expression byCD27� naive and CD27� memory B cells from patientswith primary SS and healthy control subjects wasanalyzed using flow cytometry, single-cell reversetranscriptase–polymerase chain reaction (RT-PCR),and migration assays.

Results. In contrast to healthy subjects, signifi-cantly higher expression of both surface CXCR4 andCXCR4 messenger RNA (mRNA) was seen in peripheralblood B cells from patients with primary SS. Thesedifferences were most prominent in CD27� naive Bcells (P < 0.0006). In addition, significantly higherfrequencies of CD27� naive B cells from patients withprimary SS expressed mRNA for the inhibitory regula-tor of G protein signaling 13 (P � 0.001). Expression ofCXCR5 by peripheral CD27� memory B cells wasmoderately diminished in patients with primary SScompared with healthy controls (P � 0.038). No signif-icant differences were noted in the expression ofCXCR3, CCR6, CCR7, and CCR9 between B cells fromhealthy controls and those from patients with primarySS. Transmigration assays of blood B cells from pa-

tients with primary SS and healthy controls showedcomparable responses of CD27� naive B cells butsignificantly diminished responses of activated primarySS CD27� memory B cells to the ligands of CXCR4 andCXCR5, CXCL12 (P � 0.032), and CXCL13 (B lympho-cyte chemoattractant; B cell–attracting chemokine 1;P � 0.018), respectively, when compared with thosefrom healthy controls. Finally, compared with controls,peripheral reduction but glandular accumulation ofCXCR4�,CXCR5�,CD27� memory B cells was identi-fied in patients with primary SS.

Conclusion. In primary SS, overexpression ofCXCR4 by circulating blood B cells does not translateinto enhanced migratory response to the cognate ligand,CXCL12. This migratory response may be modulated byintracellular regulators. Retention of CXCR4�,CXCR5�,CD27� memory B cells in the inflamed glands seems tocontribute to diminished peripheral CD27� memory Bcells in primary SS.

Primary Sjogren’s syndrome (SS) is characterizedby chronic focal lymphocytic inflammation of the lacri-mal and salivary glands, resulting in keratoconjunctivitissicca and xerostomia. Both interaction of activated glan-dular epithelial cells with infiltrating lymphoid and den-dritic cells and systemic lymphocyte derangement arethought to contribute to the pathogenesis of primary SS(for review, see refs. 1 and 2). The lymphoid infiltrateswithin the inflamed glands often contain germinal center(GC)–like structures consisting of T and B cell aggre-gates with proliferating lymphocytes and a network offollicular dendritic cells and activated endothelial cells(3,4). Besides antigen-driven clonal proliferation of Bcells (3,5), analyses of inflamed glandular tissue frompatients with primary SS also reveal a polyclonal accu-mulation of CD27� memory B cells and CD27high

plasma cells (6,7). Moreover, immunophenotyping stud-ies indicate that there is disturbed B cell homeostasis in

Supported by the DFG (grants Do 491/4-7 and the SFB421/project C7).

1Arne Hansen, MD, Karin Reiter, Till Ziprian, Annett Jacobi,MD, Andreas Hoffmann, Mirko Gosemann, Jurgen Scholze, MD,Thomas Dorner, MD: Charite University Hospital, Berlin, Germany;2Peter E. Lipsky, MD: National Institute of Arthritis and Musculo-skeletal and Skin Diseases, NIH, Bethesda, Maryland.

Address correspondence and reprint requests to Arne Han-sen, MD, Department of Medicine, Outpatient Department, Univer-sity Hospital Charite, Schumannstrasse 20/21, 10098 Berlin, Germany.E-mail: arne.hansen@charite.de.

Submitted for publication June 29, 2004; accepted in revisedform March 30, 2005.

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patients with primary SS, with diminished frequencies andabsolute numbers of peripheral CD27� memory B cells(6,8,9). More recently, a single-cell messenger RNA(mRNA) study showed further abnormalities, especially inthe mechanisms of heavy chain switch recombination (10).

Chemokines and their corresponding chemokinereceptors play an important role in lymphopoiesis, dif-ferentiation, homing, recirculation, and immune re-sponses of lymphocyte subsets under physiologic andpathologic conditions (11–14). The inflamed glands seenin primary SS have been shown to express a uniqueprofile of adhesion molecules, cytokines, and chemo-kines, including overexpression of CXCL13 (B lympho-cyte chemoattractant [BLC]; B cell–attracting chemo-kine 1 [BCA-1]) mRNA and protein, a centralchemokine involved in B cell homing (15–17), as well asof CCL19, CCL18, CXCL9 (monokine induced byinterferon-�), and CXCL10 mRNA (17,18,19). More-over, CXCR5-expressing B cells have been detected inthe glandular infiltrates of patients with primary SS(15,16). Thus, it has been proposed that disturbances inchemokine expression may selectively guide and regu-late lymphoid subsets into or within the target tissues aswell as the (re)circulation between blood and secondarylymphoid organs of patients with primary SS.

In order to delineate these disturbances ingreater detail and to determine whether these abnormal-ities might contribute to the disturbed B cell homeostasisin patients with primary SS, we analyzed the expressionof chemokine receptors known to provide critical posi-tioning clues for B cells and plasma cells during devel-opment and/or immune responses, including CXCR3,CXCR4, CXCR5, CCR6, CCR7, and CCR9 (11,12,20).

PATIENTS AND METHODS

Patients. After the local ethics committee grantedapproval and the patients provided informed consent, hepa-rinized whole blood samples (10 ml) were obtained from 21patients with primary SS (20 women; mean � SD age 57.6 �14.6 years, age range 25–79 years, 1 man; age 44 years) at theDepartment of Medicine, University Hospital Charite, Berlin.The mean � SD disease duration was 7.1 � 3.8 years (range1–13 years). The patients fulfilled both the American Collegeof Rheumatology (21) and the revised American–EuropeanConsensus Group (22) classification criteria for primary SS.All patients tested positive for antinuclear antibodies (finespeckled pattern) as well as for anti-Ro and/or anti-La anti-bodies and/or rheumatoid factor. All had focal lymphocyticsialadenitis of the minor salivary glands (focus score �1/4 mm2)and a positive Schirmer I test result. The patients received noglucocorticoids or immunosuppressive drugs. As controls, hep-

arinized blood samples from apparently healthy subjectsand patients with systemic lupus erythematosus (SLE),matched by age and sex with the primary SS patients were alsoanalyzed.

Peripheral blood mononuclear cells (PBMCs) wereobtained by centrifugation on Ficoll-Paque (Amersham Phar-macia Biotech, Uppsala, Sweden) gradients, as previouslydescribed (23). In addition, PBMCs were also analyzed, mono-nuclear cells were prepared, as previously described (6,7), fromminor salivary gland biopsy samples from 4 patients withprimary SS and 1 female patient with nonspecific sialadenitis.

Fluorescence-activated cell sorting. For flow cytomet-ric analysis of chemokine receptor expression on peripheralCD19�,CD27� naive and CD19�,CD27� memory B cells,PBMCs from 16 patients with primary SS, 10 healthy controlsubjects, and 12 SLE patients were stained with a fluoresceinisothiocyanate (FITC)–conjugated monoclonal antibody(mAb) to CD19 (clone HD37; Dako, Glostrup, Denmark),with a Cy5-labeled mAb to CD27 (clone 2E4; a kind giftfrom Dr. Rene van Lier, Department of Immunobiology,Academic Medical Center, Amsterdam, The Netherlands),and with phycoerythrin (PE)–labeled mAb specific for oneof the following chemokine receptors: CXCR3 (clone 1C6;BD PharMingen, San Diego, CA), CXCR4 (clone 12G5; BDPharMingen), CXCR5 (FAB 190F; R&D Systems, Minne-apolis, MN), CCR6 (clone 11A9; BD PharMingen), CCR7(FAB 197F; R&D Systems), or CCR9 (FAB 179F; R&DSystems). PE-conjugated IgG2a (clone G155-178; BDPharMingen) and IgG2b (clone 133303; R&D Systems) (asnegative controls) were used in conjunction with the respec-tive chemokine receptor–specific antibodies. Incubation withantibodies was performed in phosphate buffered saline(PBS)/0.5% bovine serum albumin (BSA)/5 mM EDTA at 4°Cfor 15 minutes. Subsequently, cells were washed twice in PBS/2% BSA/4 mM EDTA. Propidium iodide (1 �g/ml; Sigma,Munich, Germany) was added immediately before flow cyto-metric analysis to exclude dead cells. Flow cytometric analyseswere performed using a FACSCalibur and CellQuest software(Becton Dickinson, San Jose, CA). For analysis of CXCR4 andCXCR5 coexpression, streptavidin–peridin chlorophyllprotein–labeled/biotinylated anti-CD19 (clone 1D3; BDPharMingen) and FITC-labeled anti-CXR5 (FAB 190F; R&DSystems) mAb were used in combination with anti-CXCR4and anti-CD27 mAb (shown above).

Single-cell reverse transcriptase–polymerase chain re-action (RT-PCR). Altogether, 720 single-sorted CD19�,CD27� and CD19�,CD27� B cells from 4 patients withprimary SS (168 CD27� naive cells, 168 CD27� memorycells) and 4 healthy controls (192 CD27� naive cells, 192CD27� memory cells) were analyzed. Individual B cells weresorted (FACSVantage; Becton Dickinson) into single wellscontaining modified 1� RT-PCR buffer (5 mM dithiothreitol,400 ng oligo[dT]18, 0.2 mM dNTP, 1% Triton X-100, 10 unitsRNasin, 40 units avian myoblastosis virus reverse transcrip-tase), as previously described (10,24). First-strand complemen-tary DNA (cDNA) was generated at 42°C for 60 minutes.Transcripts for the chemokine receptors CXCR3, CXCR4,CXCR5 splice variant 1, CXCR5 splice variant 2, and theinhibitory regulator of G protein signaling 13 (RGS13) (25)were amplified by specific nested PCR protocols using 5 �lcDNA in the first round and 5-�l aliquots of the external PCR

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mixtures in the second round. GAPDH-specific transcriptswere analyzed as internal controls. The PCR conditions in-cluded a 5-minute denaturation at 94°C, followed by 35 cyclesof denaturation at 94°C for 1 minute, annealing for 45 seconds,and extension at 72°C for 1 minute. Oligonucleotide sequencesare shown in Table 1. The PCR products were separated on1.2% agarose gel. Following column purification, several PCRproducts from all primer combinations were directly se-quenced using the BigDye Termination Sequencing kit (PerkinElmer, Emeryville, CA) and analyzed with an automatedsequencer (ABI 377; Perkin Elmer). Sequence alignmentswere performed by BLASTN searches against nucleotidedatabases (National Center for Biotechnology Information,Bethesda, MD; online at www.ncbi.nlm.nih.gov/blast). To cal-culate the sensitivity of each specific nested PCR protocol(e.g., for CXCR4, CXCR5, or GAPDH), limiting dilutionexperiments with purified target DNA were performed, whichindicated that as few as 1–10 cDNA copies could be detectedwith each of the nested protocols used.

Transmigration assay. CD� peripheral blood Bcells were enriched by positive immunomagnetic separation(Miltenyi Biotec, Bergisch Gladbach, Germany) and subse-quently incubated overnight at 37°C under 5% CO2–bufferedconditions in RPMI 1640 medium (Biochrom, Berlin, Ger-many) supplemented with 2 mM L-glutamine, 10% fetal calfserum, 25 mg/ml penicillin/streptomycin, and 1 �g/ml lipopoly-saccharide (LPS) (from Escherichia coli; Sigma). Subsequently,cell migration was examined in wells containing transwell inserts(Costar, Bodenheim, Germany) with a 6.5-mm diameter and5-�m pores using fibronectin (Invitrogen, Karlsruhe, Germany)–

precoated membranes, as previously described (26,27). Briefly,5 � 105 B cells per upper well were suspended in RPMI 1640medium supplemented with 0.5% BSA (Sigma) and then incu-bated for 90 minutes at 37°C under 5% CO2–buffered conditions.Migrated and nonmigrated cells from each patient were analyzedseparately by flow cytometry for the expression of CD19 andCD27. Optimal chemokine concentrations for migration were50 nM for CXCL12 and 250 nM for CXCL13. In addition, thetransmigratory capacity of peripheral B cells was also analyzedwithout preincubation. B cells from 5 patients with primary SSand 5 healthy controls were analyzed.

Statistical analysis. Data are expressed as the mean �SD. Statistical analysis was performed using GraphPad Prism3.0 software for Windows (GraphPad Software, San Diego,CA). Frequencies of B cells were calculated using CellQuestsoftware, and variations in the chemokine receptor expressionon B cells were compared using the nonparametric Mann-Whitney U test. Fisher’s exact test was used to comparedifferences in the frequencies of cells expressing chemokinereceptor– or RGS13-specific mRNA transcripts, respectively.P values less than 0.05 were considered significant.

RESULTS

Analysis of chemokine receptor expression byperipheral blood B cells using flow cytometry. Using4-color flow cytometry, CD19� B cells were analyzedfor the expression of CD27 as a marker of memory B

Table 1. Oligonucleotides used for specific nested polymerase chain reaction protocols*

Oligonucleotide Sequence (5� 3 3�) NCBI accession no. Position Product size, bp

CXCR3-F CTC-CCA-GAC-TTC-ATC-TTC-CTG-TC NM_001504 618–640 453CXCR3-R CAA-GAG-CAG-CAT-CCA-CAT-CC 1051–1070CXCR3-FN CCA-CCC-ACT-GCC-AAT-ACA-AC 667–686 378CXCR3-RN CGG-AAC-TTG-ACC-CCT-ACA-AA 1025–1045CXCR4-F GAA-CCA-GCG-GTT-ACC-ATG-GA AF348491 29–48 785CXCR4-R ATG-TAG-TAA-GGC-AGC-CAA-CA 794–813CXCR4-FN TAA-CTA-CAC-CGA-GGA-AAT-GGG-C 73–94 588CXCR4-RN ACC-ATG-ATG-TGC-TGA-AAC-TGG-A 639–660CXCR5-F/V1 GAG-CCT-CTC-AAC-ATA-AGA-CAG-TGA-CCA X68149 31–57 948CXCR5-R/V1 GCC-ATT-CAG-CTT-GCA-GGT-ATT-GTC NM_001716 955–978CXCR5-FN/V1 CGC-TAA-CGC-TGG-AAA-TGG-AC 95–114 310CXCR5-RN/V1 GCA-AAG-GGC-AAG-ATG-AAG-ACC 384–404CXCR5-F/V2 ACC-TCC-AAG-AGA-GCT-AGG-GTT-CC NM_032966 123–145 925CXCR5-R/V2 GCC-ATT-CAG-CTT-GCA-GGT-ATT-GTC 1024–1047CXCR5-FN GGT-CTT-CAT-CTT-GCC-CTT-TGC 453–473 266CXCR5-RN TGG-CGA-AGA-GAA-TCT-CTG-GCA-A 697–718RGS13-F ATG-AGC-AGG-CGG-AAT-TGT-TGG-A NM_002927 282–303 477RGS13-R GAA-ACT-GTT-GTT-GGA-CTG-CAT-A 737–758RGS13-FN GGT-CCA-GTA-GTC-TAT-GCA-GCA-T 411–431 223RGS13-RN AGT-GGG-TTC-CTG-AAT-GTT-CCT-G 611–632GAPDH-F TGA-AGG-TCG-GAG-TCA-ACG-GAT NM_002046 86–106 740GAPDH-R TTC-TAG-ACG-GCA-GGT-CAG-GTC-C 804–825GAPDH-FN CCT-TCA-TTG-ACC-TCA-ACT-ACA-TGG-T 182–206 469GAPDH-RN GAG-GGG-CCA-TCC-ACA-GTC-TT 631–650GAPDH-FN2 ATC-ACC-ATC-TTC-CAG-GAG-CGA 295–315 308GAPDH-RN2 GTC-ATG-AGT-CCT-TCC-ACG-ATA-CCA 579–602

* NCBI � National Center for Biotechnology Information; F � forward; R � reverse; N � nested; V1 � splice variant 1; V2 � splice variant 2;RGS13 � regulator of G-protein signaling 13.

CHEMOKINE RECEPTORS ON B CELLS IN PRIMARY SS 2111

cells and for the expression of chemokine receptorCXCR3, CXCR4, CXCR5, CCR6, CCR7, or CCR9.Dead cells were excluded by propidium iodide staining.Frequencies of positive cells and the geometric meanfluorescence intensity of anti–chemokine receptor stain-ing were calculated according to statistical thresholds setin reference to staining with negative control antibodies.The frequency of peripheral CD27�,CD19� memory Bcells was significantly reduced in patients with primarySS compared with healthy control subjects (mean � SD13.3 � 12.3% versus 25.6 � 7.2%; P � 0.0014), whereasthe frequency of CD19�,CD27� naive B cells wassignificantly enhanced in patients with primary SS(mean � SD 86.1 � 12.0% versus 74.4 � 7.2%; P �0.0014) as reported previously (6,8,9).

To ensure that these alterations in patients withprimary SS did not further influence the analyses, eitherCD19�,CD27� or CD19�,CD27� B cells were gated,and the chemokine receptor expression was subse-quently analyzed within each subpopulation (Figures 1Aand B). Significantly higher percentages of CXCR4-expressing CD27� naive B cells (mean � SD 95.2 �2.9% versus 87.7 � 4.2%; P � 0.0003) and CXCR4-

expressing CD27� memory B cells (78.5 � 10.1% versus63.6 � 17.8%; P � 0.0251) were found in patients withprimary SS compared with healthy controls. Moreover,the geometric mean fluorescence intensity of anti-CXCR4 staining on CD27� naive B cells (mean � SD189.5 � 75.8 versus 95.1 � 30.4; P � 0.0021) and CD27�memory B cells (62.6 � 26.1 versus 28.7 � 14.6; P �0.0021) was significantly enhanced in patients with pri-mary SS as compared with healthy controls (Figures 2Aand B).

To evaluate whether this alteration is specific forprimary SS or is a general feature of systemic auto-immune diseases, peripheral blood B cells from SLEpatients were also analyzed for surface expression ofCXCR4. The frequency of CXCR4�,CD27� naive Bcells (mean � SD 95.2 � 2.9 in primary SS versus 84.5 �9.5 in SLE; P � 0.0017) and CD27� memory B cells(78.5 � 10.1 in primary SS versus 52.0 � 14.5 in SLE;P � 0.0001) was significantly enhanced in patients withprimary SS as compared with SLE patients, whereasthere were no significant differences between SLE pa-tients and healthy subjects. The density of CXCR4expression on CD27� naive B cells (geometric mean

Figure 1. Comparison of the frequencies of A, CXCR3�, CXCR4�, and CXCR5� peripheral B cells, and B, CCR6�, CCR7�, and CCR9�peripheral B cells from patients with primary Sjogren’s syndrome (SS) and healthy control subjects, as determined by flow cytometry.CD19�,CD27� or CD19�,CD27� B cells were gated, and chemokine receptor expression of each subpopulation was analyzed separately.Significant differences between patients with primary SS (pSS) and normal healthy subjects (NHS) are indicated. In addition, the following weresignificantly different by Mann-Whitney U test: in healthy controls, CD27�,CXCR3� versus CD27�,CXCR3�, CD27�,CXCR5� versusCD27�,CXCR5� (P � 0.0001 for both), and CD27�,CXCR4� versus CD27�,CXCR4� (P � 0.0007); in patients with primary SS,CD27�,CXCR3� versus CD27�,CXCR3�, CD27�,CXCR4� versus CD27�,CXCR4� (P � 0.0001 for both), and CD27�,CXCR5� versusCD27�,CXCR5� (P � 0.0013). Bars indicate the median.

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fluorescence intensity �SD 189.5 � 75.8 in primary SSversus 85.3 � 78.6 in SLE; P � 0.0015) and CD27�memory B cells (62.6 � 26.1 in primary SS versus 19.3 �12.9 in SLE; P � 0.0001) of patients with primary SS wasfound to be significantly enhanced compared with thosein patients with SLE. Again, there were no significantdifferences in CXCR4 expression between SLE patientsand healthy controls. Notably, the density of CXCR4expression was significantly higher on CD27� naive Bcells than on CD27� memory B cells in all 3 groupsanalyzed (healthy controls, and patients with primary SSand SLE; P � 0.0007 for all comparisons) (Figure 2A).

The frequency of CXCR5-expressing CD27�memory B cells (mean � SD 79.6 � 14.8% in patientsversus SD 89.8 � 4.1% in controls; P � 0.043) (Figure1A) and the density of CXCR5 expression on CD27�memory B cells (geometric mean fluorescence intensity� SD 259.6 � 159.4 in patients versus 388.9 � 60.4 incontrols; P � 0.038) were significantly diminished inpatients with primary SS as compared with healthycontrols. No further differences in chemokine receptorexpression on blood B cells between patients with pri-mary SS and healthy controls were identified, neither inthe CXCR5 expression on CD27� B cells nor in theexpression of CXCR3, CCR6, CCR7, and CCR9 onCD27� or CD27� B cells.

Experiments were performed to examine thecellular distribution and chemokine receptor expressionby B cells in salivary glands of patients with primary SS.Comparison of peripheral and glandular B cells from 4patients with primary SS revealed an accumulation ofCD27� memory B cells in minor salivary gland infil-trates. The vast majority of these glandular CD27�memory B cells expressed both CXCR4 and CXCR5 (anexample is shown in Figure 3B). Conversely, analysis ofperipheral CD27� memory B cells from patients withprimary SS revealed a markedly diminished proportionof CXCR4�,CXCR5� cells as compared with healthycontrols (Figure 3A). In contrast, there was no reductionof peripheral CXCR4�,CXCR5�,CD27� naive B cellsin patients with primary SS compared with healthycontrols (data not shown).

Amplification of chemokine receptor transcriptsfrom individual B cells by single-cell RT-PCR. ThecDNA samples from all individual cells sorted in thecurrent study were tested for their integrity by amplifi-cation of the “housekeeping” gene GAPDH. Each of thesubsets manifested a comparable high frequency ofpositive cells (mean � SD 46.4 � 2.5% in healthysubjects versus 46.1 � 7.7% in patients with primary SS).Notably, a significantly enhanced frequency of CD27�naive B cells that expressed CXCR4 transcripts was

Figure 2. Analysis of the geometric mean fluorescence intensity (MFI) of CXCR4 surface expression in patients with primary Sjogren’s syndrome(SS) and in healthy control subjects. A, Comparison of CXCR4 expression by peripheral CD27� naive and CD27� memory B cells from patientswith primary SS (pSS), normal healthy subjects (NHS), and patients with systemic lupus erythematosus (SLE), determined by flow cytometry. Valuesare the difference in geometric MFI ( MFI) compared with an appropriate negative control antibody. Significant differences between primary SSpatients and healthy controls as well as between primary SS patients and SLE patients are indicated. In addition, CXCR4 density ( MFI) wassignificantly different between CD27� naive and CD27� memory B cells in healthy subjects (P � 0.0001), in primary SS patients (P � 0.0001), andin SLE patients (P � 0.0007) by Mann-Whitney U test. Bars indicate the median. B, Density of CXCR4 expression (geometric [G] mean) onperipheral CD27� naive B cells from a healthy subject (solid histogram) and a primary SS patient (open histogram).

CHEMOKINE RECEPTORS ON B CELLS IN PRIMARY SS 2113

found in patients with primary SS (60 of 168 cells;35.7%) compared with healthy controls (26 of 144 cells;18.1%) (P � 0.0006) (Figures 4A and B). Furthermore,in patients with primary SS, the frequency of CXCR4-transcript–positive B cells was significantly enhanced inCD27� naive B cells (60 of 168 cells; 35.7%) comparedwith CD27� memory B cells (37 of 168 cells; 22.0%)(P � 0.0079). A significantly increased percentage ofCD27� memory B cells expressing CXCR4-specific

mRNA transcripts was also found in patients withprimary SS (37 of 168 cells; 22.0%) compared withhealthy controls (33 of 240 cells; 13.8%) (P � 0.033).

Both known CXCR5–mRNA splice variants(variant 1 NM_001716 and variant 2 NM_032966; Na-tional Center for Biotechnology Information database[28,29]) were analyzed in healthy controls and in pa-tients with primary SS. It was found that individualperipheral B cells expressed either variant 1 (which

Figure 3. CXCR4 and CXCR5 coexpression on CD27� memory B cells in patients with primary Sjogren’s syndrome (SS) and in healthy controls.A, CD19�,CD27� memory B cells from the peripheral blood of 3 patients with primary SS (pSS) and of 3 normal healthy subjects (NHS) wereanalyzed by flow cytometry according to their coexpression of CXCR4 and CXCR5. B, Flow cytometric analysis of peripheral blood and glandularCD19� B cells from a patient with nonspecific sialadenitis (control) and a patient with primary SS assessed for the coexpression of CD27.CD19�,CD27� memory B cells from the primary SS patient were further gated and analyzed for their coexpression of CXCR4 and CXCR5. Dataare representative of results from 4 primary SS patients. Gates were set according to isotype controls.

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encodes a protein that is 45 amino acids longer at theN-terminus than isoform 2) or variant 2. However, it iscurrently not known whether there is a functional dif-ference between the variants. Importantly, no differ-ences in CXCR5–mRNA expression were found be-tween patients with primary SS and healthy subjects.Finally, when the expression of mRNA transcripts forthe chemokine receptor–signaling regulator proteinRGS13 (25) was examined, a significantly enhancedpercentage of CD27� naive B cells expressing RGS13transcripts was found in patients with primary SS (28 of168 cells; 16.7%) compared with healthy controls (7 of144 cells; 4.9%) (P � 0.001), whereas the portion ofCD27� memory B cells expressing RGS13 mRNA inpatients with primary SS was not significantly differentfrom that in healthy controls (Figure 4A).

Migration of CD27� naive and CD27� memoryB cells in vitro. To demonstrate the functionality ofchemokine receptor expression, peripheral CD19� Bcells from 5 patients with primary SS and 5 healthysubjects were analyzed using transmigration assays. Nosignificant differences in response to either CXCL12 or

CXCL13 were found between patients with primary SSand healthy controls when unstimulated B cells wereanalyzed (Figure 5A). However, both in patients withprimary SS and in healthy controls, the transmigratorycapacity of B cells was significantly enhanced by LPSstimulation (P � 0.0001). After stimulation, significantlyhigher percentages of CD27� memory B cells than ofCD27� naive B cells migrated in response to CXCL12and CXCL13 in both groups. Of note, there weresignificantly diminished responses of CD27� memory Bcells from patients with primary SS to both CXCL12(mean � SD 76.8 � 7.8% versus 86.0 � 3.8% incontrols; P � 0.032) and CXCL13 (76.6 � 7.2% versus88.0 � 1.8% in controls; P � 0.018), respectively, ascompared with those from healthy subjects (Figure 5B).

DISCUSSION

Recent studies have shown disturbances in peri-pheral B cell populations in primary SS, with signifi-cantly enhanced CD27� naive and diminished CD27�memory B cells (6,8,9). This was confirmed in the

Figure 4. Single-cell reverse transcriptase–polymerase chain reaction (RT-PCR) analysis in patients with primary Sjogren’s syndrome (SS) andhealthy controls. A, Frequencies of CXCR3, CXCR4, CXCR5, and regulator of G protein signaling (RGS13) mRNA transcript–positive individualCD19�,CD27� naive B cells and CD19�,CD27� memory B cells in 4 patients with primary SS (pSS) and 4 normal healthy subjects (NHS). Thepercentages of individual B cells from which a clear distinct product was obtained with the respective nested RT-PCR, in relation to the total sortedindividual B cells, are shown. The specificity of each nested RT-PCR protocol was confirmed by DNA sequencing. Values are the mean and SEM.Significant differences as determined by Fisher’s exact test are indicated. B, Example of CXCR4-specific nested RT-PCR products on 1.2% agarosegel from individual CD27� memory and CD27� naive B cells from a healthy subject and a patient with primary SS. M � DNA marker.

CHEMOKINE RECEPTORS ON B CELLS IN PRIMARY SS 2115

present study. An accumulation of CD27� memory Bcells in inflamed tissue (6,10), altered recirculation of Bcell subsets from these sites (7), and/or altered B celldifferentiation (30) may contribute to these distur-bances. The underlying assumption of the present studywas that the expression of chemokine receptors onperipheral B cells might reflect a distinct B cell patternin primary SS, with specific functional consequences.Overall, a differential expression of chemokine recep-tors by peripheral blood B cells from patients withprimary SS was identified.

First, there was overexpression of CXCR4 byblood B cells from patients with primary SS that wasmost prominent in CD27� naive B cells. In particular,significantly higher frequencies of CXCR4-expressing Bcells were detected in patients with primary SS com-pared with healthy controls, both in CD27� naive Bcells (P � 0.0003) and in CD27� memory B cells (P �0.0251). Moreover, the density of CXCR4 surface ex-pression was significantly enhanced in patients withprimary SS as compared with healthy controls (P �0.0021) for both CD27� naive and CD27� memory Bcells. Remarkably, these differences were also evidentwhen blood B cells from patients with primary SS werecompared with those from patients with SLE (P �

0.0015 for CD27� naive cells and P � 0.0001 forCD27� memory cells), whereas there was no significantdifference in CXCR4 expression between healthy sub-jects and SLE patients.

Thus, this abnormality appeared to be specific toprimary SS rather than being common in systemicautoimmunity. Moreover, when individual B cells wereanalyzed for chemokine receptor mRNA, significantlyenhanced frequencies of CD27� naive B cells (P �0.0006) and CD27� memory B cells (P � 0.033) ex-pressing CXCR4 transcripts were found in patients withprimary SS compared with healthy controls. However,CXCR4 overexpression by blood B cells from patientswith primary SS did not translate into an enhancedmigratory response to the corresponding chemokine,CXCL12, as compared with those from healthy controls.These results suggest that there was intracellular mod-ulation of the migratory response in primary SS B cells.

To assess the discrepancy between CXCR4 ex-pression and migratory response to the correspondingchemokine (CXCL12) in greater detail, mRNA expres-sion of RGS13 (25,31) as one potential influencingfactor was examined in individual CD27� naive andCD27� memory B cells. RGS13 belongs to the family ofRGS proteins (for review, see refs. 25 and 31) that are

Figure 5. Transmigration assays showing the frequencies of in vitro–migrated peripheral CD19�,CD27� naive and CD19�,CD27� memory Bcells from 5 patients with primary Sjogren’s syndrome (SS) and 5 normal healthy subjects in response to either 50 nM CXCL12 or 250 nM CXCL13(B lymphocyte chemoattractant; B cell–attracting chemokine 1). A, Unstimulated B cells and B, lipopolysaccharide-stimulated B cells from patientswith primary SS (pSS) and healthy controls (NHS). Values are the mean and SEM. Significant differences between CD19�,CD27� naive andCD19�,CD27� memory B cells as determined by Mann-Whitney U test are indicated.

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thought to be responsible for the fine-tuning of theintracellular signaling of G protein–coupled receptors,especially chemokine receptors. Thereby, they establishthresholds for responsiveness, provide stop signals formigration, and/or contribute to receptor desensitizationto corresponding chemokines (25,31). RGS13 has re-cently been shown to modulate signaling throughCXCR4 and CXCR5 in murine and human germinalcenter B cells possessing one of the most limited pat-terns of expression of known RGS (25). Moreover,cotransfection with RGS13 inhibited the migrationalresponse of CXCR4-transfected Chinese hamster ovarycells toward CXCL12 in vitro (25). In the present study,significantly enhanced expression of RGS13 mRNA byCD27� naive blood B cells from primary SS patients(P � 0.001) was found. Thus, the combined data suggestthat CXCR4 overexpression by blood B cells frompatients with primary SS might be partly compensatedby up-regulation of the inhibitory regulator proteinRGS13 and, thereby, might contribute to the discrep-ancy between CXCR4 expression and migratory responseto its corresponding ligand, CXCL12.

In this context, it is well established that surfaceexpression of chemokine receptors does not necessarilyindicate their migratory functionality (32–34). Indeed,the responsiveness of chemokine receptors for theirrespective ligands is differentially regulated (e.g., byRGS proteins) during the orchestration of the migrationof lymphoid subpopulations into anatomic compart-ments, their development, activation, and immune re-sponse (26,27,31–36). B cells from different develop-mental stages, e.g., developing bone marrow B cells (36),B cells leaving GC structures (33), and medullary plas-mablasts leaving lymph nodes (34), have been found toexpress high levels of surface CXCR4 but were unre-sponsive to CXCL12. In this regard, there is someevidence that CXCR4 might fulfill additional functionsbesides chemotaxis, e.g., cell growth, proliferation, andtranscriptional activation (11,33,37,38). In accordance,CXCL12 treatment has been found to increase NF-�Bactivity in nuclear extracts from CXCR4-transfectedmurine pre–B lymphoma cells (37). Moreover, it hasbeen shown that CXCL12–CXCR4 interaction stimu-lates G protein–mediated activation processes in peri-pheral T cells (39). Although it is currently unclearwhether CXCR4 also fulfills such additional functions inhuman blood B cells, it might be speculated that CXCR4and RGS13 (over)expression might contribute to or,alternatively, reflect abnormal B cell stimulation inprimary SS, which warrants further studies.

Compared with healthy controls, flow cytometric

analysis revealed a moderately diminished frequency ofCXCR5�,CD27� memory B cells (P � 0.0425) com-bined with a lower density of CXCR5 surface expressionon CD27� memory B cells (P � 0.038) in patients withprimary SS. In this context, the CXCL13–CXCR5 pair-ing has been shown to be critically involved in thehoming of B cells into lymphoid follicles, as well as in thedevelopment of organized lymphoid follicles(28,29,40,41). The formation of ectopic lymphoid tissuein chronic inflammatory disease, such as primary SS, is acomplex process regulated by an array of cytokines,adhesion molecules, and chemokines (4,13), partly mim-icking signals found in normal lymphoid organogenesis(42). Whether expression of CXCL12 and CXCL13 inthe target tissues of patients with primary SS is closelyassociated with the development of GC-like structuresor, rather, is a feature of the entire inflammatory processis still controversial (4,15).

However, it has been suggested that CXCL13overexpression in the inflamed glands of patients withprimary SS plays an active role in the recruitment oflymphoid cells as infiltrating cells, mostly B cells, whichexpress the cognate receptor CXCR5. Thus, in patientswith primary SS, overexpression of CXCL13 in inflamedglands with consequent local retention of CXCR5-bearing B cells (15,16) might also lead to reducedfrequencies of peripheral CD27� memory B cells ex-pressing lower levels of surface CXCR5. This assump-tion has been supported by recent studies of primary SSindicating accumulation of memory B cells in glandularinfiltrates (6,10). In accordance with this, simultaneousanalyses in this study of B cells from peripheral bloodand minor salivary gland infiltrates of patients withprimary SS also revealed an accumulation of CD27�memory B cells in the inflamed glands. The vast majorityof these infiltrating CD27� memory B cells coexpressedCXCR5, along with CXCR4. Conversely, diminishedfrequencies of peripheral blood CD27� memory B cellscoincide with a striking reduction of the peripheralCXCR4�,CXCR5� memory B cell subpopulation inpatients with primary SS. Thus, glandular coexpressionof both CXCL12 and CXCL13 (15–18) seems to navi-gate this subpopulation of peripheral CD27� memory Bcells into the inflamed glands, where it resides. Consis-tent with this, residual circulating peripheral CD27�memory B cells from patients with primary SS showed adiminished migratory response to the correspondingligands of CXCR4 and CXCR5, CXCL12 and CXCL13,respectively, after stimulation. This suggests that mem-ory B cells with less migratory capacity remain in theblood as a result of the selective migration and retention

CHEMOKINE RECEPTORS ON B CELLS IN PRIMARY SS 2117

of CXCR4�,CXCR5� memory B cells into the in-flamed glands.

In conclusion, peripheral B cells in primary SSmanifest specific abnormalities in chemokine receptorexpression and function of both memory and naivesubpopulations. The abnormally expressed receptors,CXCR4 and CXCR5, specifically bind the chemokines,CXCL12 and CXCL13 (BLC; BCA-1), respectively,which are important for navigating lymphocytes in lym-phoid tissues, and, thereby, for lymphocyte homeostasis(11,12,42). Migration/retention of CXCR4�,CXCR5�,CD27� memory B cells in the inflamed target tissues ofpatients with primary SS appears to account for thediminished number of these cells in the peripheralblood. However, the increased number of naive B cellsin the peripheral blood does not appear to reflect analteration in chemotaxis. Rather, the increased expres-sion of CXCR4 appears to be offset by intracellularmodulation with resultant normal migratory responsive-ness. Both differences might reflect an abnormality inactivation status of the naive subpopulation. Thus, dis-turbed B cell differentiation, activation, and/or (re)cir-culation between immune compartments may contributeto the disturbed B cell homeostasis in primary SS(10,30). Detailed understanding of the impact of chemo-kines and their cognate receptors, including their regu-lation, may allow the development of future therapeuticinterventions in primary SS, a disease unresponsive toclassic immunosuppression.

ACKNOWLEDGMENT

We are grateful to Thoralf Kaiser for excellent techni-cal assistance.

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