decreased expression of signal-transducing cd3 ζ chains in t cells from the joints and peripheral...

Decreased Expression of Signal-Transducing CD3z Chains inT Cells from the Joints and Peripheral Blood of RheumatoidArthritis Patients

M. MATSUDA*, A.-K. ULFGREN†, R. LENKEI‡, M. PETERSSON*, A. C. OCHOA§, S. LINDBLAD†,P. ANDERSSON¶, L. KLARESKOG† & R. KIESSLING*,**

*Microbiology and Tumor Biology Center (MTC) at Karolinska Institute;†Department of Rheumatology, Karolinska Hospital; and‡Calab, Stockholm, Sweden;§NCI, Frederick, Washington, USA;¶Dana Farber Cancer Institute, Boston, MA, USA; and**Department of Experimental Oncology, Radiumhemmet, Karolinska Hospital, Stockholm, Sweden

(Received 19 September 1997; Accepted in revised form 18 November 1997)

Matsuda M, Ulfgren A-K, Lenkei R, Petersson M, Ochoa AC, Lindblad S, Andersson P, Klareskog L,Kiessling R. Decreased Expression of Signal-Transducing CD3z Chains in T Cells from the Joints andPeripheral Blood of Rheumatoid Arthritis Patients. Scand J Immunol 1998;47:254–262

Although T cells from patients with rheumatoid arthritis (RA) have previously been determined to have poorproliferative responses to a variety of stimuli, the underlying mechanism is not known. We have investigatedthe expression of the signal-transducingz molecule in subsets of T cells and natural killer (NK) cells derivedfrom the peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) of RApatients using quantitative flow cytometry, Western blot analysis and immunohistochemistry. A decrease ofz

expression was apparent in all investigated lymphocyte subsets from the PBMC and SFMC of RA patients, ascompared to the corresponding subsets from healthy age- and sex-matched controls. A less pronouncedreduction of cell surface-located CD3e, CD4 and CD8 was also located in T cells from SFMC as compared toPBMC from RA patients. Biochemical demonstration of the low or absent CD3z in PBMC from patients withRA was achieved by Western blot analysis. Immunohistochemical staining and image analysis also confirmedthe low expression ofz chains in synovial tissue of RA patients. The possibility that the decreased expressionof z and of immune functions of T cells from RA patients may be related to the presence of free oxygenradiclals, as we have previously reported in cancer patients, should be considered.

Rolf Kiessling, Karolinska Institute, Microbiology and Tumor Biology Center, S-17177 Stockholm, Sweden

INTRODUCTION

Rheumatoid arthritis (RA) is characterized by a chronic inflam-matory process involving the synovial membrane and leading tothe degradation of the articular cartilage and the subchondralbone [1, 2]. It is believed that the manifestations of the diseaseare the results of an immunological process within the affectedjoints, and the synovial tissue of RA patients is infiltrated by avariety of immunological cells including T and B lymphocytes[1, 3]. Experimental and clinical evidence for T-cell involvementin the pathology of the disease exists, pointing to a localdysregulation of T-cell functions in the inflamed joint [4].

Although T cells from peripheral blood and synovial fluid ofRA patients express increased levels of activation markers,including HLA-DR [5–9], they are functionally impaired.Thus, a poor response to the polyclonal mitogens PHA and

pokeweed mitogen (PWM) in autologous mixed lymphocytereactions and to stimulation with antibodies to the CD3 complexof the T-cell receptor (TCR), as well as a decreased production ofinterleukin 2 (IL-2) following activation, have been described inT cells from RA patients [9–13]. Taken together, these observa-tions point to a defect in signal transduction via the TCR–CD3structure of T cells from RA patients. More direct evidencefavouring this interpretation has also been published, demon-strating that cross-linking of CD3 molecules on RA patients’T-cell surfaces induced a two- to threefold lower Ca2þ influxresponse as compared to controls [14].

In order to analyze the molecular basis for the defective TCR-CD3 signal transduction in T cells from RA patients, we havefocused this study on the CD3z molecule. The cytoplasmicdomain of this 16-kD molecule, which is present primarily as adisulphide-linked homodimer, is involved in signal transduction

Scand. J. Immunol.47, 254–262, 1998

q 1998 Blackwell Science Ltd

and subsequent activation of T cells [15–17] and has beenimplicated in the assembly of the CD3 complex [18]. Similarto the situation in RA patients, cancer patients have T cells withreduced proliferative capacity [19], a defect in their CD3z

expression and in other signal-transducing molecules [20–22].Since alterations in the CD3z chain may disrupt the internalsignalling of the TCR, leading to deficient or altered T-cellactivation, this suggests a new way of understanding the mechan-isms responsible for deficient T-cell responses in RA patients.We here describe a markedly decreased expression of thez

molecule in T cells and natural killer (NK) cells from synovialfluid and tissue as well as from the peripheral blood of patientswith RA.

MATERIALS AND METHODS

Patients. Patients with active rheumatoid arthritis (defined accordingto the 1987 American College of Rheumatology Criteria, or ACR, forRA) were recruited from the open care unit at the Department ofRheumatology, Karolinska Hospital, Stockholm, Sweden. Peripheralblood as well as synovial fluid was obtained in heparinized vials andtransported to the laboratory within 30 min. Synovial biopsies wereobtained by surgical synovectomy. All patients received non-steroid anti-inflammatory drugs, but no other anti-rheumatic drugs. Age-matched andsex-matched controls were recruited from healthy staff of the Departmentof Rheumatology and its research unit or from healthy blood donors.

Isolation of cells. Peripheral blood mononuclear cells (PBMC) wereisolated from heparinized blood of healthy donors and RA patients byFicoll–Paque (Pharmacia, Uppsala, Sweden) density gradient centrifu-gation. Synovial fluid mononuclear cells (SFMC) of RA patients wereisolated as previously described [23].

Flow cytometry. Three-colour immunofluorescence staining and flowcytometric analysis was performed as previously described [24]. Mono-clonal antibodies (MoAbs) conjugated with fluorescein isothiocyanate(FITC), phycoerythrin (PE) or peridinin chlorophyll protein (PerCP)were purchased from Becton-Dickinson (San Jose, CA, USA). TheCD45RO MoAb was purchased from Ortho (Ortho DiagnosticsSystem Inc., Raritan, NJ, USA).

Determination of antibody binding capacity (ABC) was performedwith Quantum Simply Cellular (QSC) beads produced by Flow CytometryStandards Corporation (FCSC: Puerto Rico, USA). The QSC beads havea mixture of four highly uniform microbead populations of the same sizewhich are coated with goat anti-mouse antibodies and have varyingcapacities to bind mouse MoAbs. Included in the mixture is a blankpopulation of microbeads which has no specific binding capacity formouse immunoglobulins. The accompanying software regresses thebinding capacities of the microbeads (stained with fluorochrome-con-jugated MoAbs) against their corresponding peak channels. The regres-sion curve allows estimation of the number of MoAb molecules bound totarget cells [24]. A total of 20ml QSC beads produced by FCSC wasadded to 50ml phosphate-buffered saline (PBS) containing the necessaryamount of MoAb (established by previous titration and usually 20–40ml). The samples were processed further according to the method usedfor lymphocyte staining (direct from CD antigens and indirect forz

chain), including the fixation step. The mean fluorescence intensity(MFI) of the respective antigen, when analyzed on the whole populationof positive lymphocytes or on separate subsets, was translated by meansof the software into molecules of antibody bound to the cell membrane.

The expression ofz molecules was investigated by flow cytometric

analysis of permeabilized cells, using the MoAb TIA-2 specific for theintracellular part of thez chain [25]. Isolated SFMC or PBMC werestabilized by fixation with 0.5% formaldehyde in PBS for 20 min on iceand then permeabilized by the addition of digitonin (10mg/ml) for 5 minon ice. After confirming that the permeabilization was complete byTrypan Blue uptake, cells were pelleted and resuspended in PBScontaining saturating concentrations (1–2mg/sample) of purified anti-z(TIA-2, IgG1) or control immunoglobulin IgG1 (X-931; Dakopatts,Glostrup, Denmark) at the same concentration. After 20 min on ice,cells were washed twice using PBS containing 0.05% Tween-20, andthen further incubated with anti-mouse FITC (FL1) as described [25] foran additional 20 min on ice. After blocking with normal mouse serum(NMS) and washing three times with ice-cold PBS/Tween-20, cells weredouble-stained with PE–FL2-conjugated MoAbs to CD3 (Beckton-Dickinson) and then subjected to flow cytometric analysis using aFACScan flow cytometer (Becton-Dickinson). Cells which were CD3þ

were gated and measured for Mean FL1 (Mean Fluorescence 1) valuesmeasuringz-chain staining of the gated cell population.

Handling of biopsies. Biopsies were immediately snap frozen in dryice with Isopentan in OCT-compound (Tissue Tek : Miles Inc., Eikhart,USA). All tissues were kept at¹708C until sectioned.

Immunohistochemistry and image analysis. Two different immuno-histochemical staining protocols were used. In the first protocol (Table 1),6–8-mm thick cryostat sections were mounted on gelatine-coated glassslides (Novakemi, Stockholm, Sweden) and slides were frozen at¹708C.Slides were fixed in cold 2% PBS-buffered formaldehyde directly fromthe freezer. The initial step of the tissue staining was blocking ofendogenous peroxidase activity by 1% hydrogen peroxide and 2%sodium nitride dissolved in Earl’s Balanced Salt Solutions (EBSS)with Ca2þ and Mg2þ (Gibco Ltd, Paisley, UK) supplemented with0.01M Hepes buffer and 0.1% saponin (Reidal de Haen, AG, Seelze,Germany), hereafter termed EBSS–saponin solution, for 1 h at roomtemperature in the dark. After three additional washes in EBSS–saponin,the slides were incubated overnight at room temperature in a humidchamber with 100ml of anti-CD3e (Becton-Dickinson) or anti-z (Mono-zeta 1: Biomira, Cranbury, NJ, USA) in EBSS–saponin. Control stainingwas performed in parallel with species- and isotype-matched myelomaproteins. Slides were washed three times with EBSS–saponin andincubated with 1% normal goat serum in EBSS–saponin for 15 min atroom temperature, in order to reduce background caused by IgG–Fc

Low Expression of CD3z Chains in T Cells from Rheumatoid Arthritis255

q 1998 Blackwell Science Ltd,Scandinavian Journal of Immunology, 47, 254–262

Table 1. Immunohistochemical staining of CD3z and CD3e insynovial tissue of RA patients

Percentage of stained areaa withMoAb specific for

Patientb CD3 e CD3 z

1 27.7 1.42 11.7 5.73 17.7 1.34 2.5 1.2

aShows the percentage of stained area, as determined on frozensections stained with an anti-CD3e MoAb or anti-CD3z MoAb without‘retrieving’ method.

b Performed on synovial tissue from four different RA patientssubjected to synovectomy.

interactions by the biotinylated secondary antibodies. Secondary anti-bodies were biotin-goat-anti-mouse IgG1 (Caltag Lab, San Francisco,CA, USA) and incubation time was 30 min at room temperature. Afterthree washes with EBSS–saponin, EAP complex was added for 60 minat room temperature. After one last wash with only EBSS the reactionwas developed by 0.5 mg/ml diaminobenzidine (DAB: Vector Lab).Counterstaining was with Mayers Haematoxylin for 1–5 min and theslides were mounted with 1/9 (v/v) of PBS–glycerol.

In the second immunohistochemical protocol (Fig. 2), in which theTIA-2 MoAb was used, the antigens were ‘retrieved’ to optimize immuno-reactivity, as previously described [26]. Sections 7mm thick weremounted on gelatine-coated glass slides and air dried. Endogenousperoxidase activity was blocked by incubation in 0.3% H2O2/methanolfor 30 min. Tissue sections were immersed in sodium citrate buffer(0.1M pH 6.0) and boiled for 6 min in a microwave oven at highestpower, followed by a 20-min period of cooling down. The slides werewashed in EBSS–0.1% saponin three times. Before the first incubationof primary antibody, a blocking step was included with 2% normal goatserum for 15 min in EBSS–saponin, in order to reduce backgroundcaused by IgG–Fc interactions by the biotinylated secondary antibodies.After three additional washes in EBSS–saponin, the slides were incubatedovernight at room temperature in a humid chamber with 100ml of theanti-CD3 e polyclonal (Dakopatts) or anti-z TIA-2 monoclonal anti-bodies in EBSS–saponin. The stained sections were examined using aPolyvar 2 microscope (Reichert-Jung, Vienna, Austria). Numbers of Tcells were assessed as the number of CD3e-staining cells within theentire tissue section that was investigated. The tissue sections wereanalyzed with a Quantimet 570 (Q570) image analyzer (Leica Cam-bridge Ltd, Cambridge, UK). The image processor was directed by a PCwith special software [27]. The results of the image analysis wereexpressed as per cent positively stained area within the entire tissuesection under investigation.

Biochemical analysis ofz-chain expression. Five million PBMC weredirectly lysed into hot SDS sample buffer to avoid the effect of protease.Then the DNA was disrupted by centrifugation in the presence of glassbeads. Samples were run in a 10% SDS PAGE under non-reducingconditions, transferred to nitrocellulose and probed with the TIA-2MoAb [25].

RESULTS

Distribution of lymphocyte subsets in the peripheral blood andsynovial fluid of RA patients

The composition of various lymphocyte subsets in the PBMC

and SFMC of RA patients, and in peripheral blood of age- andsex-matched healthy controls, was analyzed (Table 2). Extendingearlier observations [28], there was an increased percentage of Tcells (CD3þCD16¹) in SFMC as compared to PBMC in RApatients. This increase of T cells in the SFMC was due to arelative increment of CD8þ cells, resulting in a decreased CD4/CD8 ratio. The T cells in the SFMC to a large extent expressedHLA-DR and CD45RO, indicating that they were activatedmemory cells. A lower frequency of cells expressing the lowaffinity FcR for IgG (FcgRIIIA a, CD16) in SFMC as comparedto PBMC was recorded, whether expressed on NK cells(CD3¹CD16þ) or T cells (CD3þCD16þ). There were alsofewer B cells (CD20þ) in the SFMC was compared to PBMCof RA patients.

The lymphocyte subset composition of PBMC from RApatients was not significantly different compared to that ofPBMC from controls, with the exception of a somewhat lowerpercentage of CD8þCD45ROþ cells in the PBMC of RA patientsas compared to controls (Table 2).

Analysis of the expression ofz in T-cell and NK-cell subsetsfrom PBMC and SFMC of RA patients by quantitative flowcytometry

The relative density ofz expression was measured by quantita-tive flow cytometry [24] and defined as antibody bindingcapacity (ABC) in subsets of T cells and NK cells derivedfrom the PBMC and SFMC of 12 RA patients and from 10healthy age-matched controls. A decrease ofz expression wasapparent in lymphocytes and in a number of different subsets ofthese from the PBMC of RA patients, as compared to thecorresponding subset from the PBMC of controls (Table 3).All lymphocyte subsets were equally low inz expression, withthe exception that NK cells, defined as CD3¹/CD16þ, had ahigher z expression as compared to T cells (CD3þ/CD16¹) inPBMC of patients and controls. The presence of CD16 in theCD3þ population (CD3þCD16þ) also gave a higherz expressionas compared to the CD3þCD16¹ subset.

Although z expression was lower in the SFMC-derivedas compared to PBMC-derived lymphocytes for all subsetsinvestigated, this difference was not significant except for theCD8þCD28¹ subset (Table 3).

Analysis of the expression of surface CD molecules in T cellsfrom SFMC and PBMC of RA patients by quantitative flowcytometry

The expression of a panel of lymphocyte surface CD antigenswas analyzed quantitatively in PBMC and SFMC from RApatients and controls (Table 4). Since CD3z is known to affectthe assembly of the CD3 molecular complex [18], we investi-gated if cell surface expression of CD3 would also be reduced onT cells from RA patients. The SFMC of the patients had asignificantly reduced cell surface expression of CD3e, as com-pared to PBMC of the same patients (Table 4). The suppression

256 M. Matsuda et al.


Fig. 1. Western blot analysis ofz expression in PBMC of RA patientsand controls. Cell lysates from 5×106 PBMC derived from twohealthy controls (control PBMC) and five RA patients (RA PBMC)were run in a 10% SDS PAGE under non-reducing conditions,transferred to nitrocellulose and probed with the TIA-2 MoAb.

of CD3 e in the SFMC was more pronounced in the CD8 ascompared to the CD4-subset of T cells. This decrease of CD3e

was restricted to the surface-located pool of this molecule, as thetotal intracellular CD3e expression, as measured in permeabil-ized cells, was unaltered (data not shown). A slightly increasedexpression of surface CD8 was afferent in the SFMC as com-pared to PBMC of the patients.

Although there was a reduced expression of CD3e whencomparing PBMC of patients with those of controls, this differ-ence was not significant (Table 4). An unexpected finding wasthat T cells from patients expressed significantly fewer CD4 andCD8 molecules, known to be of importance in TCR activation[29, 30]. This did not reflect a generally lower expression of cellsurface molecules on lymphocytes of the patients, as the expres-sion of CD5, CD20, CD28 and CD45 was not altered.

Biochemical analysis of the TCR complex in T cells fromPBMC of RA patients

A biochemical analysis ofz expression in PBMC of RA patientswas performed. Cell lysates of PBMC from five RA patients wererun by SDS PAGE under non-reducing conditions, transferred tonitrocellulose and probed with the anti-z MoAb. Three of theanalyzed samples revealed a total absence of the 30-kD bandcorresponding to thez:z homodimer, while two showed the

presence of thez chains, although at a lower intensity than thetwo PBMC samples from healthy individuals included as con-trols (Fig. 1). This analysis therefore confirms that PBMC fromRA patients had decreased expression of thez chains. In additionto thez:z homodimer, lysates from PBMCs contained an approxi-mately 24-kD protein that was recognized by anti-z. This bandprobably representsz:g heterodimers of CD3z and FceRIgpresent in both T cells and NK cells.

Image analysis of immunohistochemical staining for CD3z

and CD3e on synovial tissue from RA patients

The expression of CD3z and CD3 e on T cells within thesynovial tissue from RA patients was analyzed by immuno-histochemistry of tissue and staining was evaluated by imageanalysis. In accordance with previous data, considerable differ-ences were observed in the number of CD3e between biopsiesfrom different patients as well as between different areas of thesame biopsy.

Both of the two different anti-CD3z MoAbs used for theimmunohistochemical analysis gave very weak staining of syno-vial tissue from RA patients (Fig. 2), although areas with a highernumber of positive cells could be detected. A markedly highernumber of cells positive for CD3e compared to CD3z wasapparent in most investigated areas (Fig. 2 and Table 1). There



Table 2. Distribution of lymphocyte subsets in PBMC and SFMC of RA patients

Medians percentages of positive lymphocytes

A. Controlsa B. RA Bloodc C. RA SFMCe

Subset n¼ 10 P A/Bb n¼ 12 P B/Cd n¼ 12

1. CD3þ f 73.1 (70.1–79.4) NS 77.7 (72.0–81.0) 0.0001 90.7 (88.8–93.1)2. CD3þCD16þ 0.9 (0.4–2.2) NS 0.8 (0.3–1.6) 0.0102 0.2 (0.1–0.4)3. CD3þCD16¹ 71.7 (67.1–77.5) NS 74.7 (70.8–77.0) 0.0001 89.2 (86.5–90.8)4. CD3¹CD16þ 13.8 (5.3–16.6) NS 10.2 (6.2–12.1) 0.0001 1.5 (0.8–2.9)5. CD4þ 44.5 (34.2–50.2) NS 47.6 (34.4–50.6) NS 42.7 (28.9–56.0)6. CD8þ 25.1 (15.8–29.5) NS 21.2 (15.5–26.3) 0.0404 41.8 (29.5–51.3)7. CD4/CD8 ratio 2.2 (1.2–2.8) NS 2.0 (1.7–2.5) 0.0404 1.02 (0.60–1.90)8. CD20 3.5 (2.4–6.4) NS 6.5 (2.6–10.6) 0.0243 0.9 (0.4–1.6)9. CD4þHLA-DRþ 3.9 (2.4–4.4) NS 3.0 (2.2–5.3) 0.0003 17.7 (10.3–22.8)

10. CD8þHLA-DRþ 4.0 (3.2–5.8) NS 2.8 (1.4–5.1) 0.0007 27.1 (13.5–32.1)11. CD4þCD45ROþ 20.6 (17.0–29.7) NS 22.6 (18.9–26.8) 0.0306 31.5 (28.4–49.1)12. CD8þCD45ROþ 13.3 (10.1–15.3) 0.0046 6.8 (5.8–8.2) 0.0051 36.0 (18.7–52.2)13. CD4þCD28þ 37.2 (32.9–45.5) NS 42.1 (35.2–50.7) NS 45.6 (30.4–58.2)14. CD8þCD28þ 14.5 (13.6–17.3) NS 12.6 (10.7–15.6) 0.0002 34.0 (21.5–43.8)

aPBMC from age-matched and sex-matched healthy controls.b P values derived from comparing values in column A with those in column B with a Mann–Whitney non-

parametric test.c PBMC from RA patients.d P values derived from comparing values in column B with those in column C with a Mann–Whitney non-

parametric test.eSFMC from RA patients.f Analyzed on permeabilized cells with CD3–PE MoAb.

was also, however, a notable variation in the proportion of CD3z

and CD3e stainings in the different specimens and even withindifferent parts of the same specimen. Strong staining of tissuesections from regional lymph nodes, included as a positivecontrol, were consistently observed with both of the anti-CD3z MoAbs (Fig. 2).

DISCUSSION

Alterations in signal-transducing molecules, including CD3z,have been described in lymphocytes from tumour-bearing miceand cancer patients [20–22, 31]. We here report that this phenom-enon can also occur in an autoimmune condition, which mayexplain the defective TCR–CD3 signal transduction previouslyobserved in T cells from RA patients [14]. Alterations in signal-transducing molecules may therefore be a common feature of

conditions characterized by sustained chronic inflammation,including chronic bacterial infections [32]. Several featuresrelating to the defect inz expression described here are similarto previous findings in mice and humans with tumours. Thus, theobservedz low phenotype was ‘global’ among all studied subsetsof T cells and NK cells from RA patients, as we previouslyobserved in patients with colorectal carcinomas [33]. This wouldargue against thez-low phenotype being a result of induction ofanergy in a defined subset of T-cell clones for example, but beingmore compatible with the effect of a mechanism acting on alllymphocytes.

Previous studies have reported a local down-regulation ofz

expression in tumours, using flow cytometry and immunohisto-chemistry [20–22, 33, 34]. The local suppression of CD3z

expression in the joints of RA patients was studied by immuno-histochemical analysis and image analysis of synovial tissue.



Table 3. Analysis of the expression ofz in T cell and NK cell subsets from PBMC of RA patients and controlsby quantitative flow cytometry

Median ABCz chain 1/103 molecules (P25–P75)a

Lymphocyte A. Controlsb B. RA Bloodd C. RA SFMCf

subsets n¼ 10 P A/Bc n¼ 12 P B/Ce n¼ 12

1. All lymphocytes 26.0 (16.0–30.7) 0.0017 10.8 (5.8–15.6) NS 5.8 (5.1–11.9)2. CD4þ 26.8 (21.1–33.6) 0.0011 10.6 (4.5–16.5) NS 6.2 (5.1–17.1)3. CD8þ 21.5 (16.5–27.0) 0.0022 10.0 (4.6–13.4) NS 5.2 (4.1–10.6)4. CD3þCD16þ 44.4 (39.2–69.8) 0.0102 24.0 (13.3–47.5) NS 16.9 (10.5–22.4)5. CD3þCD16¹ 26.6 (20.0–31.7) 0.0004 8.6 (4.6–14.6) NS 6.4 (5.7–13.2)6. CD3¹CD16þ 56.4 (40.2–66.6) 0.0030 25.5 (16.2–45.1) NS 12.0 (8.4–16.6)7. CD3þCD56þ 26.9 (20.5–29.5) 0.0004 8.2 (4.3–15.6) NS 6.0 (4.8–13.2)8. CD4þHLA-DRþ 34.4 (23.5–37.3) 0.0009 9.3 (5.1–16.1) NS 7.7 (5.7–16.4)9. CD4þHLA-DR¹ 28.0 (22.6–34.6) 0.0009 9.2 (4.4–14.7) NS 5.7 (4.2–14.4)

10. CD8þHLA-DRþ 25.8 (15.1–30.1) 0.0005 8.4 (5.6–12.0) NS 5.5 (4.3–12.9)11. CD8þHLA-DR¹ 23.1 (18.5–30.8) 0.0005 9.6 (4.8–13.6) NS 5.1 (3.4–12.7)12. CD4þCD38þ 25.9 (20.3–32.8) 0.0007 11.1 (5.4–16.9) NS 8.0 (5.9–16.2)13. CD4þCD38¹ 26.0 (18.6–34.3) 0.0010 9.8 (4.4–16.0) NS 6.1 (4.9–15.3)14. CD8þCD38þ 20.7 (17.1–29.6) 0.0024 9.1 (6.4–14.0) NS 6.2 (5.1–11.3)15. CD8þCD38¹ 19.3 (15.1–27.8) 0.0024 5.3 (4.5–13.2) NS 4.8 (3.9–9.0)16. CD4þCD45ROþ 22.5 (13.8–28.5) 0.0343 10.4 (4.7–17.4) NS 12.9 (6.8–18.2)17. CD4þCD45RO¹ 24.5 (13.3–30.8) 0.0199 12.5 (5.1–17.8) NS 9.5 (5.2–12.5)18. CD8þCD45ROþ 18.6 (12.0–24.3) 0.0262 8.5 (4.5–14.1) NS 9.9 (4.9–14.0)19. CD8þCD45RO¹ 19.8 (12.8–27.3) 0.0199 10.9 (5.9–16.0) NS 7.1 (3.8–10.0)20. CD4þCD28þ 28.0 (20.8–34.4) 0.0022 10.3 (4.6–17.7) NS 6.8 (5.4–15.2)21. CD4þCD28¹ 17.8 (13.2–34.9) 0.0161 9.2 (4.2–14.7) NS 5.3 (4.2–9.8)22. CD8þCD28þ 22.5 (15.7–26.1) 0.0014 7.8 (3.6–14.0) NS 6.3 (4.3–10.5)23. CD8þCD28¹ 25.1 (17.1–33.1) 0.0022 11.5 (6.2–15.7) 0.0127 6.1 (4.0–9.6)

aThe median antibody binding capacity (ABC)z chain 1/103 molecules (P25–P75).b PBMC from age-matched and sex-matched healthy controls.c P values derived from comparing values in column A with those in column B with a Mann–Whitney non-

parametric test.d PBMC from RA patients.eP values derived from comparing values in column B with those in column C with a Mann–Whitney non-

parametric test.f SFMC from RA patients.

Areas with close to normalz expression were occasionallydetected, however, demonstrating that a considerable micro-variation in z expression exists in the synovia. Since thismethod does not require anyin-vitro isolation or processing oflymphocytes, and the biopsies used were snap frozen and storedin liquid nitrogen, the possibility that the observed reduction inz

expression is anin-vitro artefact, i.e. due to enzymatic degrada-tion of this molecule by endopeptidase A released from granu-locytes [35], seems unlikely. Collectively, these results arecompatible with the notion that the mechanism leading todecreased expression ofz is active at the site of inflammation.

Two non-receptor protein tyrosine kinases (PTKs) have beenimplicated in TCR functions; the p59fyn, associated with theTCR–CD3 and p56lck, associated with CD4 and CD8, both ofwhich belong to thesrc-family PTKs [29, 30]. Furthermore, aphysical association between CD4 and TCR complexes has beendemonstrated by co-immunoprecipitation of CD3e with the CD4lck complex [29]. In T cells from cancerous mice and patients,expression of both these tyrosine kinases were reduced [21, 22,31]. It is of interest that we describe herein that the expressionof CD4 and CD8 is also reduced in SFMC and PBMC fromRA patients, as we have previously reported in patients withcolorectal carcinomas [33]. Our data therefore demonstrate thatthe altered composition and down-regulation of components ofthe CD3–TCR complex also applies to the CD4 and CD8co-receptors. Several different tyrosine-dependent signal trans-duction pathways may therefore be impaired in lymphocytes

from RA patients and cancer patients. The finding of unalteredexpression of CD5 or CD28 on T cells from the patients,however, demonstrates that not all molecules involved inT-cell activation are expressed less in T cells from RA patients.

No correlation between the patient’s serum C-reactive proteinor soluble CD8 value and the CD3z expression in PBMC wasrecorded, when analyzed with serum samples taken on threeoccasions with 1-month intervals from 10 of the RA patientsfrom the present study (data not shown). We found no evidencethat the observed alterations in the TCR–CD3 complex arerelated to medication or the age of the patient (data notshown). Since we suggest that the observed alterations of theTCR–CD3 complex may be directly related to the previouslypublished defect in the proliferating capacity of T cells from RApatients, it is of interest to note that this defect was also deter-mined to be independent of the drug treatment and age of thepatients [10]. Similarly, it was also concluded that the impairedCD3-activated Ca2þ mobilization responses in T cells from RApatients were unrelated to the duration of the disease [14].

We could confirm earlier observations [5–8] of a high percen-tage of T cells expressing HLA-DR and CD45RO in the SFMCof RA patients. The presence of a high frequency of activatedmemory T cells in the joints of RA patients therefore appears togo hand-in-hand with reduced function and, as demonstratedhere, decreased expression of signal-transducing TCR-associatedmolecules. We have also described a high frequency of activatedmacrophages in the RA joint [9].



Table 4. Analysis of the expression of surface CD molecules in T cells from PBMC and SFMC of RA patientsby quantitative flow cytometry

Median ABC CD antigens 1/103 molecules (P25–P75)a

Lymphocyte A. Controlsb B. RA bloodd C. RA SFMCf

subsets n¼ 10 P A/Bc n¼ 12 P B/Ce n¼ 12

1. CD3–FITC 122.9 (91–141) NS 97.4 (83–118) 0.0017 73.5 (64–82)2. CD3–FITC (CD4þ) 130.2 (112–148) NS 108.0 (92–133) 0.0303 86.9 (70–101)3. CD3–FITC (CD8þ) 103.5 (84–129) NS 90.2 (75–107) 0.0022 65.7 (56–73)4. CD4–PerCP 71.1 (56.7–76.8) 0.0378 58.5 (52.0–63.6) NS 63.1 (55.5–65.9)5. CD8–PE 179.1 (149–210) 0.0033 114.5 (91–151) 0.0404 150.1 (144–162)6. CD5–FITC 45.6 (39.9–50.9) NS 45.9 (37.5–51.3) NS 43.0 (30.3–56.0)7. CD20–PerCP 31.6 (26.1–40.8) NS 32.2 (22.2–43.9) 0.0600 51.1 (35.9–69.1)8. CD28–PE 7.0 (6.2–7.8) NS 6.4 (4.6–7.6) NS 5.6 (4.6–7.7)9. CD28–PE (CD4þ) 7.9 (6.6–9.1) NS 7.5 (5.4–9.0) NS 7.4 (6.1–9.5)

10. CD28–PE (CD8þ) 5.9 (4.7–9.4) NS 5.6 (2.9–7.6) NS 4.6 (3.6–6.0)11. CD45–FITC 61.2 (40–106) NS 76.1 (67.7–91.9) NS 90.8 (69–113)

aThe median antibody binding capacity (ABC)z chain 1/103 molecules (P25–P75).b PBMC from age-matched and sex-matched healthy controls.c P values derived from comparing values in column A with those in column B with a Mann–Whitney non-

parametric test.d PBMC from RA patients.eP values derived from comparing values in column B with those in column C with a Mann–Whitney non-

parametric test.f SFMC from RA patients.

We have recently suggested that the mechanism underlyingthe impaired expression ofz in T cells and NK cells from patientswith cancer may be related to the release of free oxygen radicalsby activated macrophages derived from the tumour lesion [36].Thus, splenic macrophages from tumour-bearing mice or macro-phages isolated from metastatic lesions of human melanomas, oractivated normal macrophages but not residual macrophages,induced the loss of CD3z and other structural components of theCD3 complex [37, 38]. This was accompanied by loss of tumour-specific cytotoxicity and mobilization of intracellular Ca2þ flux,and the mechanism responsible involved the secretion of H2O2

[36]. The role of activated macrophages suppressingz expression

through a mechanism related to oxidative stress was also subse-quently reported in a mouse experimental tumour system [39].Therefore, the possibility that reactive oxygen metabolites pro-duced by macrophages/monocytes or possibly granulocytes con-tribute directly to alterations in functions and in signal-transducingmolecules of lymphocytes in individuals with RA should also beconsidered. Arguing for this possibility, evidence for the role ofan altered redox state in the hyporesponsiveness of synovial Tcells in RA was recently published [40]. Regardless of the under-lying mechanism, assessment ofz expression based on flowcytometry of PBMC or on immunohistochemistry of tissue-located T cells, may increase our understanding of T-cell



Fig. 2. Immunohistochemical staining of frozensections of a human lymph node (A–D) and synovialtissue from an RA patient (E–H). The staining wasperformed with the protocol for ‘retrieving’ antigensby microwave oven, as described in Materials andMethods, and a magnification of×100 was used.Staining with antibodies against CD3e (B and F),CD3 z (C and G, the TIA-2 MoAb; D and H,mono-zeta 1 MoAb) and isotype-matched (IgG1)control MoAb (A and E) are shown.

function in RA and possibly also in other chronic inflammatoryconditions.

ACKNOWLEDGMENTS

This work was supported by grants from the Swedish MedicalResearch Council and from the Swedish Labour Market Insur-ance Company (AFA). We thank Dr R. A. Harris for criticalreading and linguistic advice.

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decreased expression of signal-transducing cd3 ζ chains in t cells from the joints and peripheral...

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