participation of the cd69 antigen in the t-cell activation process of patients with systemic lupus...

Participation of the CD69 Antigen in the T-Cell Activation Processof Patients with Systemic Lupus Erythematosus

J. C. CRISPIN, A. MARTIŃEZ, P. DE PABLO, C. VELASQUILLO & J. ALCOCER-VARELA

Department of Immunology and Rheumatology, Instituto Nacional de la Nutricioń Salvador Zubirań, Mexico City, Mexico

(Received 24 October 1997; Accepted in revised form 16 February 1998)

Crispin JC, Martıńez A, de Pablo P, Velasquillo C, Alcocer-Varela J. Participation of the CD69 Antigen in theT-Cell Activation Process of Patients with Systemic Lupus Erythematosus. Scand J Immunol 1998;48;196–200

Accumulating evidence has implicated T cells in the pathogenesis of systemic lupus erythematosus (SLE).The CD69 antigen is an integral membrane protein rapidly induced on the surface of activated lymphocytes.We obtained CD4þ and CD8þ T cells from normal subjects and patients with SLE. The percentage of CD69expression in freshly isolated cells and afterin-vitro incubation with mitogens was quantified by three-colourimmunofluorescent staining. Expression of this protein was increased in both CD4þ and CD8þ T-cell subsetsfrom SLE patients when compared with normal cells, although the difference was significant only in the CD8þ

T-cell subset (P¼ 0.05). Cellular activation increased CD69 expression. When stimulated with anti-CD2/CD2R or phytohaemagglutinin (PHA), the percentage and absolute numbers of CD69þ cells were lower inpatients than in controls. Addition of anti-interleukin (IL)-10 monoclonal antibody (MoAb) increased thepercentage ofin-vitro CD69 expression in SLE cells. These results suggest that the peripheral bloodlymphocytes from patients with SLE have an intrinsic defect that alters their activation process, includingthe expression of CD69, and might explain some of the T immunoregulatory abnormalities observed in thesepatients.

Jorge Alcocer-Varela MD, Departmento de Immunologıá y Reumatologıá, Instituto Nacional de la NutriciońSalvador Zubirań, Tlalpan 14000, Me´xico

INTRODUCTION

Systemic lupus erythematosus (SLE) is an autoimmune diseasecharacterized by functional alterations of both T and B lympho-cytes. At present we ignore which of the abnormalities detectedin SLE patients are primary and which are the consequence of thealtered immunoregulation. It seems likely that a defect or defectsin the mechanisms that closely regulate the activation andsubsequent proliferation of T lymphocytes (peripheral tolerance)could be responsible for the myriad of alterations observed inthese patients [1, 2].

In previous reports we have demonstrated that the activationprocess of lymphocytes of patients with SLE is impaired.Specifically, we found alterations in the autologous mixedlymphocyte reaction and in the expression of activation markers[3, 4].

Lymphoid activation follows a complex mechanism, onlypartially understood. It is the process by which a lymphocyteundergoes the differentiation that will allow it to become an

effector cell and the proliferation that will augment the numberof lymphocytes specific for the antigen that elicits the response.One of the earliest changes that appears in an activated cellpopulation is a shift in the pattern of expression of some surfacemolecules that are not found in resting cells. Thea subunit of thereceptor for interleukin (IL)-2 (CD25), transferrin receptors(CD71) and CD69 are examples. These molecules are calledcell activation markers [5].

The CD69 antigen is a type II integral membrane protein witha C-type lectin-binding domain. It is a member of the naturalkiller (NK) cell gene complex family of cell-surface receptorsand is present as a homodimer formed by the association of twopolypeptides (28 kDa and 32 kDa chains) held together by disul-phide links. The two chains result from differential glycosylationof a 22.5 kDa polypeptide, encoded by a single gene present inhumans in chromosome 12 [6]. It is constitutively expressed inCD3bright thymocytes, platelets, Langerhan’s cells and bonemarrow myeloid precursors. Lymphocytes, natural killer (NK)cells, neutrophils and eosinophils express it when stimulated. It is

Scand. J. Immunol.48, 196–200, 1998

q 1998 Blackwell Science Ltd

rapidly induced on the surface of activated lymphocytes and canbe detected as early as 3–4 h following stimulation [7–9]. Itsexpression requires p21ras activation and is inhibited by agentsthat block RNA transcription and protein synthesis [10, 11].Although the kinetics of its expression suggest it may play a rolein cell activation, its function is still unclear.

In the present report we used flow cytometry to study theexpression of CD69 in peripheral blood lymphocytes obtainedfrom patients with SLE and from normal donors. We wereable to evaluate its differential expression in each of thelymphoid subpopulations at rest and stimulated with polyclonalactivators.

MATERIALS AND METHODS

Patients. We studied 13 female patients who fulfilled at least four criteriafor the classification of SLE [12], and 10 healthy controls. Their agesranged from 17 to 63 years (mean age 34.3 years). The duration of theirdisease ranged from 0.5 to 8 years, with a mean of 6.4 years. Clinicaldisease activity was scored on a 32-point scale according to the MEX-SLEDAI activity index [13]. Eight patients had active disease; four ofthem were receiving prednisone (7.5–60 mg) or immunosuppressivedrugs (azathioprine, 50–100 mg) at the time of the study.

Cell preparation. We collected 10 ml of heparinized venous bloodfrom each patient and control. Various concentrations of mitogeniclectins (12.5ml of Phytolacca americana[Pokeweed mitogen (PWM)25mg/ml, Sigma Chemical Co., St Louis, MO, USA], 2ml of phyto-haemagglutinin (PHA) [1 mg/ml, Gibco BRL, Gaithersburg, MD, USA])or monoclonal antibodies (MoAbs) (10mL of anti-CD2/CD2R [20mg/mlBecton Dickinson Immunocytometry Systems, San Jose, CA, USA])were added to 500ml aliquots of whole blood. Samples were incubatedfor 4 h in a 378C water bath.

Peripheral blood mononuclear cell cultures. Mononuclear cells(PBMC) were isolated from eight patients and seven controls byconventional methods (Histopaque 1077; Sigma Diagnostics, St Louis,MO, USA). Basal and PHA-stimulated mononuclear cell cultures con-taining 106 cells per ml of culture medium (RPMI-1640 supplementedwith 200 U/ml penicillin G, 10mg/ml gentamicin, 0.3 mg/ml L-gluta-mine and 10% fetal calf serum) were prepared, either in the presence orabsence of 1.5mg/ml of anti-IL-10 MoAb (Diaclone, Besancon, France).After 24 h of incubation cells were recovered and stained for flowcytometric analysis as detailed below.

Three-colour immunofluorescent staining. Three-colour immuno-fluorescent staining was performed according to previously publishedmethods [14]. Briefly, 50ml of each sample were stained with 10ml of atitrated mixture of three MoAb fluorochrome conjugates for 20 min atroom temperature in the dark. Every sample included anti-CD3 (Leu 4,conjugated with cyanin-5/phycoerythrin, CY-5/PE) and anti-CD69 (Leu23, conjugated with phycoerythrin, PE) and one of the following: anti-CD4, anti-CD8 or anti-CD30 (Leu 3a, Leu 2a and Ki-1 antigens,respectively, conjugated with fluorescein isothiocyanate, FITC). Inorder to eliminate the red blood cells (except in the samples of isolatedPBMC), 500 ml of lysis solution (Simultest EMK-lymphocyte lysissolution: Becton Dickinson Immunocytometry Systems) were added toeach sample, followed by a period of 15 min in the dark at roomtemperature. Finally, samples were fixed with 500ml of 3% formalde-hyde in phosphate-buffered saline (PBS) for 16 h at 48C before flowcytometric analysis. Immunoglobulin (Ig)G1 isotype control antibody

conjugates were included to establish background fluorescence. Addi-tionally, the CD3/CD19 ratio was quantified in each patient. All theMoAbs used were obtained from Becton Dickinson ImmunocytometrySystems, except the anti-CD30 which was acquired from DAKO A/S(Glostrup, Denmark).

Flow cytometry studies. Flow cytometric analysis was performedusing a FACscan flow cytometer (Becton Dickinson ImmunocytometrySystems). Initially, cells positive for CD3–CY-5/PE (FL3) were gated,and the rest of the analysis considered only the population included inthe gate (i.e. T cells). Data were displayed as two-colour dot plots(FL1 versus FL2) to measure the proportion of activated lymphocytesubsets (CD69þ). For the analysis we used FACScan software (BectonDickinson) in a Hewlett Packard computer.

Statistical analysis. Data are expressed as mean6 SD. Statisticalanalysis was performed by the non-parametric Mann–WhitneyU-test.P-values less than 0.05 between two groups were consideredsignificant.

RESULTS

Lymphocyte subsets in patients and controls

Flow cytometric analysis was performed on the unstimulatedsamples in order to assess the distribution of the differentlymphocyte subsets in SLE patients. We identified the totallymphocyte population in the forward versus side scatter dotplot (FSC versus SSC) as a defined conglomeration distinct frommonocytes and granulocytes. Although SLE patients had a lowertotal lymphocyte count than normal donors (16296 574 versus23176 366), no difference was observed in the lymphoid subsets(CD4, CD8, CD19, CD30), neither when evaluated as absolutenumbers nor as percentages (results not shown).

Proportion of CD69þ peripheral blood T cells

We studied the proportion of unstimulated peripheral blood Tcells, CD4þ and CD8þ, positive for CD69 4 h after isolation. Asshown in Table 1, the means of both CD4þ/CD69þ and CD8þ/

CD69 and T-Cell Activation in SLE197

q 1998 Blackwell Science Ltd,Scandinavian Journal of Immunology, 48, 196–200

Table 1. Expression of CD69 on freshly isolated and activated T-cellsubsets from patients with SLE and normal controls

Mean6 SD

Cell Subset Condition SLE (n¼ 13) Controls (n¼ 10)

CD4þ

CD69þ Freshly 1.46 1.3 0.96 0.8þ PHA 3.96 4.1a 10.56 8.3

CD8þ

CD69þ Freshly 2.66 2.8b 0.996 0.5þ PHA 3.36 2.2a 8.36 3.5

aP¼ 0.01 when compared to controls.b P¼ 0.05 when compared to controls.

CD69þ were higher in SLE patients than in controls. Never-theless, the difference was statistically significant only in theCD8þ/CD69þ subset (P¼ 0.05).

Expression of CD69 after cell activation with different stimuli

The blood obtained from patients and controls was stimulatedwith anti-CD2/CD2R, PWM or PHA as described in Materials

and Methods. The percentage of cells expressing CD69 waslower on lymphocytes obtained from SLE patients in response tothe different stimuli (Table 1). Differences were statisticallysignificant in both subpopulations (CD4þ and CD8þ cells)when anti-CD2/CD2R and PHA, the strongest stimuli, wereused (Figs 1 and 2). Decreased expression of CD69 was also

198 J. C. Crispin et al.


Fig. 1. Expression of CD69 (a representativeexperiment) on CD4þ T cells. A three-colouranalysis of unstimulated (upper panels) and anti-CD2-activated (lower panels) CD3þ lymphocytes(gated in FL3) of a SLE patient (right side) andher control (left side). Cells were stained asdescribed in Materials and Methods.

Fig. 2. Percentage of resting and anti-CD2-stimulated CD4þ andCD8þ T cells that express CD69 in patients with SLE and theircontrols.

Fig. 3. Percentage of CD4 T cells positive for CD69 antigen. Cellswere incubated for 24 h in culture medium alone or with anti-IL-10 MoAb. Closed circles represent cells incubated in culture mediumalone; open circles cells, incubated with the antibody.

noted in cells stimulated with PWM, although the difference didnot reach statistical significance.

Based on the reports of high levels of cytokine IL-10 in PBMCcultures from SLE patients [15], we hypothesized that thiscytokine might contribute to the low activation-induced CD69up-regulation observed on SLE T cells. We therefore incubatedSLE PBMC in anti-IL-10 MoAb-supplemented medium. Wefound an increase of CD69 expression on both CD4 and CD8T-cell subsets, from 18 to 128% (mean 59%), and from 13 to50% (mean 24%), respectively (Fig. 3).

In six of the eight patients with active disease the percentage ofCD4þ CD69þ cells correlated with the disease activity (mea-sured with MEX-SLEDAI). However, in the two patients withthe highest activity index the expression of CD69 in the CD4 T-cell subset was even lower than in the control patients (results notshown).

DISCUSSION

The events responsible for the pathogenesis of SLE are stillunclear. The evidence accumulated so far indicates that theimmune system of these patients is in a hyperactive state. Severalreports have revealed a higher expression of cell activationmarkers in lymphocytes obtained from patients with SLE thanin normal controls. This suggests that the cells from their immunesystem are a step ahead, already stimulated (activatedin vivo),when studied in experimental conditions [4, 16]. The CD69antigen is an early lymphoid activation marker. Although nofunctional role has been assigned to it, its structure and kinetics ofexpression suggest it is involved in the lymphocyte activationprocess. In this report we studied the expression of CD69 in Tlymphocytes obtained from SLE patients and found that it is downregulated, either in stimulated or unstimulated cells.

Although we observed the expression of CD69 up-regulated infreshly isolated CD4þ and CD8þ peripheral blood lymphocytes,the difference was statistically significant only for the CD8þ

subset. This result agrees with the report by Portales-Pe´rezet al.in regard to CD69 expression in stimulated and unstimulatedSLE lymphocytes [17].

A recent report found that in SLE patients the CD69/CD3 ratioin freshly obtained cells correlates with disease activity [18]. Wefound a similar correlation with the exception of the two patientswith highest disease activity. It is possible that in patients withmoderately active disease the up-regulation of CD69 is due to thehyperactivity of the immune system. Conversely, in patientswhose disease is more active, the surface expression of thismolecule may be impaired.

In the stimulation assays, the cells from SLE patients dis-played a lower response compared to cells obtained from normaldonors. These results support previous work that demonstratesthat lymphocytes from SLE patients show a suboptimal prolif-eration in response to multiple stimuli [4]. The cells stimulatedwith anti-CD2/CD2R or PHA displayed the highest activationresponse (in both patients and controls) and it was in these that asignificant difference was observed. This was an expected result,

considering that anti-CD2/CD2R and PHA are specific andpotent T-cell activators. The addition of PWM, which activatesboth T and B lymphocytes, resulted in a weaker activation andeven though the response in the lymphocytes from patients waslower than the one in controls, the difference was not statisticallysignificant.

The fact that stimulated T cells are unable to-upregulate theirsurface expression of this molecule may represent a defectintrinsic to lymphoid function in SLE, rather than a consequenceof the bizarre immunoregulation. Evidence suggests that sus-tained protein kinase C (PKC) activation is responsible for T-cellreceptor (TCR)/CD3-mediated CD69 induction, and T cells fromSLE patients display a defective response to PKC activators [10,17]. Thus, the observed down-regulation of CD69 could be theresult of the alteration in the PKC activation pathway. Alterna-tively, it is possible that the activation status in which SLElymphocytes are obtained is a special stage in which CD69 is notpresent on the cell surface.

It has been shown that in PBMC cultures from SLE patientsthe IL-10 levels are increased [17]. In order to study thecontribution of the high IL-10 level to CD69 expression, weadded anti-IL-10 to the PBMC culture. In the cells obtained fromcontrol subjects the addition of the IL-10 blocking antibody hadno effect on CD69 expression. However, in the cell cultures fromSLE patients its presence further increased the CD69 expression(Fig. 3). The increase was observed in the cells with higher basalCD69 expression. This suggests that the IL-10 plays an inhibi-tory role compensating the T-cell hyperactivity; thus when it isneutralized the quantity of activated T cells rises even further.

The expression of CD69 has been studied in other autoimmunediseases. It has been demonstrated that in T cells obtained fromsynovial fluid of patients with rheumatoid arthritis and juvenilerheumatoid arthritis, the CD69þ subset is enlarged when com-pared to peripheral blood T cells from the same patient or tonormal controls [19, 20]. In this case, the up-regulation of theactivation marker seems to be an indicator of the high proportionof activated lymphocytes that accumulate in the affected jointsrather than the result of an intrinsic cell defect as in SLE.

Taken together, the results of this report suggest that theperipheral blood lymphocytes from patients with active orinactive SLE have an intrinsic defect that impairs their activationprocess, probably involving an alteration in the activation ofPKC. Moreover, the fact that the expression of CD69 is impaired,not only because of the poor activation response, but probably asa consequence of the same defect that underlies the activationand proliferation impairment, makes this molecule a poor indi-cator of cell activation in lupus. However, it might be rewardingto study the contribution of the disturbed immunological envir-onment to the requirements of CD69 expression and T-cellactivation in this complex disease.

ACKNOWLEDGMENTS

This study was supported by a grant from El Consejo Nacional deCiencia y Tecnologı´a , Mexico.

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