603

SUBPOPULATIONS OF PRIMED T HELPER CELLS IN RHEUMATOID ARTHRITIS

NICK MATTHEWS, PAUL EMERY, DARRELL PILLING, ARNE AKBAR, and MIKE SALMON

Objective. To analyze subsets of primed T helper cells, defined by expression of the CD45RB isoform of the leukocyte common antigen, in the blood and synovial fluid (SF) of patients with rheumatoid arthritis (RA).

Methods. Three-color immunofluorescence was used to study CD45 isoform expression by peripheral blood and SF CD4+ T cells.

Results. CD45 isoform expression in the periph- eral blood of patients with either RA or reactive arthritis did not differ from that in healthy controls. SF T cells from both RA patients and reactive arthritis patients were almost exclusively primed (CD45RO+) cells. RA SF T cells expressed very low levels of CD45RB; this is the most highly differentiated subset of primed cells. Patients with acute reactive arthritis showed higher levels of CD45RBbrighf cells in their synovial fluid.

Conclusion. The highly selected cell population in SF, representing one subset of primed cells, may relate

Presented at the loth International Inflammation Sympo- sium, Birmingham, United Kingdom, September 1991.

From the Department of Rheumatology, Birmingham Uni- versity, Birmingham, and the Department of Clinical Immunology, Royal Free Hospital, London, United Kingdom.

Supported by grants from the Arthritis and Rheumatism Council for Research.

Nick Matthews, PhD: Research Fellow, Department of Rheumatology, Birmingham University; Paul Emery, MA, MD, FRCP Senior Lecturer, Department of Rheumatology, Birmingham University; Darrell PiUing, BSc: Research Associate, Department of Rheumatology, Birmingham University; Arne Akbar, PhD: Lec- turer, Department of Clinical Immunology, Royal Free Hospital; Mike Salmon, PhD: Senior Research Fellow, Department of Rheu- matology, Birmingham University.

Address reprint requests to Mike Salmon, PhD, Depart- ment of Rheumatology, The Medical School, Birmingham Univer- sity, Birmingham, B15 2TT, United Kingdom.

Submitted for publication July 22, 1992; accepted in revised form January 26, 1993.

to the apparent functional abnormalities of cells from this site in patients with RA.

The role of T lymphocytes in rheumatoid arthri- tis (RA) is a subject of controversy (1,2). Chronically inflamed synovia are heavily infiltrated by lympho- cytes; circumstantial evidence of an active role of these cells has been demonstrated by the beneficial effects seen when they are removed (3,4). However, there is very little evidence of active T cell function within joints (for review, see ref. 1). Almost all syno- vial T cells are primed, expressing high levels of an isoform of the leukocyte common antigen termed CD45RO (5,6). Primed and activated T cells appear to enter the joint in a nonspecific manner because of their high level of expression of adhesion molecules (7). The strongly biased population that results is very likely to have a different pattern of activity compared with unselected peripheral blood cells (2).

Recent studies have shown that primed T helper cells can be divided into 2 populations, accord- ing to the expression of either high or low levels of another isoform of the leukocyte common antigen, i.e., CD45RB (8). In contrast, all virgin (CD45RA+) T cells express high levels of CD45RB. There are clear differences in function between the 2 subpopulations of primed cells. CD4SRBbrigh' cells produce higher levels of interferon-y (IFNy) than does the CD45RBd"" population (8). Furthermore, purified CD45RBd"" cells produce almost no detectable interleukin-2 (IL-2) (9).

We characterized CD4+ T helper cells from the peripheral blood and synovial fluid (SF) of patients with chronic RA, and compared the results with findings in healthy control subjects, to investigate whether synovial T lymphocytes may reflect a selected

Arthritis and Rheumatism, Vol. 36, No. 5 (May 1993)

604 MATTHEWS ET AL

population of cells. We evaluated the relevance of these observations by investigating the association of CD45RB exon expression with age, as well as by studying a small group of patients with reactive arthri- tis, a more acute condition, to assess whether the results with RA patients may reflect the chronicity of their disease.

PATIENTS AND METHODS Patients. Paired samples of heparinized peripheral

blood and SF were obtained from 10 patients with active RA of at least 3 years duration. All 10 patients had disease that met the American College of Rheumatology (formerly, the American Rheumatism Association) RA classification crite- ria (10). There were 5 men and 5 women, with an age range of 26-79 years (median 57.5). Patients were assessed using standard clinical and laboratory measures; all had active disease at the time of the study. Each of the 10 patients was being treated with nonsteroidal antiinflammatory drugs in conjunction with a second-line agent.

Five patients (3 men, 2 women) with acute reactive arthritis (duration of symptoms 2-19 months, effusions <6 weeks) were also studied, using the same protocol. The ages of these patients ranged from 22 to 64 years (median 31). The initiating infection was urogenital in 2 patients, enteric in I, and the site unconfirmed in 2. Four of the patients had multiple joint involvement.

Eighteen healthy volunteers with an age range of 21-69 years (median 45) served as controls. There were 9 men and 9 women in the control group.

Cell preparation. Peripheral blood mononuclear cells (PBMC) were separated from heparinized venous blood by density gradient centrifugation. SF was pretreated with hyaluronidase (10 unitshl; Sigma, Poole, UK) for 30 min- utes, after which SF mononuclear cells (SFMC) were iso- lated using the same procedure as for PBMC.

Monoclonal antibodies. Leu3a (Becton Dickinson, Oxford, UK) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), or biotin was used to label CD4. PE-conjugated 2H4 (Coulter, Hialeah, FL) was used to identify CD45RA, PD7/26-FITC (a gift from Dr. D. Mason) was used to identify CD45RB, and biotinylated UCHL-1 (a gift from Professor P. C. L. Beverley) was used to identify CD45RO. Biotin conjugates were detected using Streptavidin- conjugated allophycocyanin from Becton Dickinson.

Flow cytometry. Analysis of CD45 isoform expres- sion within the CD4+ T cell subset was performed using 3-color immunofluorescence with a Coulter EPICS Elite flow cytometer. Paired aliquots of PBMC and SFMC were incu- bated with combinations of monoclonal antibodies. The optimal concentration of each reagent was determined by preliminary titration. Fluorescein and phycoerythrin were excited at 488 nm using an Ar laser, and allophycocyanin was excited at 633 nm with an HeNe laser.

All samples were labeled with anti-CD4, together with combinations of antibodies specific for CD45RA,

Table 1. Proportion of CD4+ T cells expressed as a fraction of total lymphocytes, and expression of CD45RA and CD45RO by CD4+ cells, in healthy control subjects and patients with arthritis*

Source CD4 CD45RA CD45RO

Healthy controls (n = 18) PBMC 44 rt 11 43 rt 11 51 ? 9

Rheumatoid arthritis PBMC 51 t 10 51 rt 11 48 t 7 patients (n = 10) SFMC 47 k 17 3 ? 3 97 rt 3

Reactive arthritis PBMC 46 k 5 52 ? 16 48 rt 17 patients (n = 5) SFMC 55 rt 12 9 rt 5 91 rt 5

* Values are the mean k SD percent. PBMC = peripheral blood mononuclear cells; SFMC = synovial fluid mononuclear cells.

CD45RB, and CD45RO. Conjugated irrelevant mouse anti- bodies of all isotypes (Coulter) were used to establish the specificity of staining. Cytometer calibration was standard- ized using fluorospheres (Immunocheck and Standardbrite; Coulter), and fluorescence compensation was adjusted using samples of cells that were labeled individually with anti-CD4 conjugated to each fluorochrome. All samples were gated on forward and side scatter to exclude dead cells. CD45 isoform expression was analyzed within the population gated as CD4+. All CD45RA+ cells are CD45RBbriph'; the division between CD45RBbngh' and CD45RBdU" populations within the CD45RO+ primed fraction was defined by the lower limit of expression within the CD45RA+ fraction.

Statistical analysis. Data were analyzed using the Mann-Whitney U test and Spearman's rank correlation.

RESULTS Relationship of CD45 isoforms to age and sex.

The expression of CD45RA, CD45RB, and CD45RO was measured in CD4+ T helper cells from the 18 healthy control subjects ranging in age from 21 to 69 years. Within this group, no correlation was found between expression of any CD45 isoform and age (r = -0.086 for CD45RA, 0.146 for CD45RB, and 0.12 for CD45RO). Similar results were obtained in the 10 patients with RA (r = -0.1 15, -0.011, and -0.045 for CD45RA, CD45RB, and CD45R0, respectively). The control group of 9 men and 9 women showed no sex-related differences in isoform expression.

Expression of CD45RA and CD45RO by CD4+ T cells in the blood and SF of patients with RA. T helper cells in peripheral blood were approximately equally divided into CD45RA-t and CD45RO+ populations. No differences were found between healthy controls and patients with RA in expression of either isoform. However, in RA SF, the vast majority of cells were of the primed CD45RO-t phenotype (Table 1) .

PRIMED T HELPER CELL SUBSETS IN RA 605

CD4+

CD4SRA+

CD45RO+

CD45RB

CD4SRB

CD45RB

NONE

CD4+

CD4+ + z

CD4+ $ U

A B C

RELATIVE FLUORESC E9 C E

CD45RB Figure 1. Populations of CD4+ T helper cells and of virgin (CD4+CD45RA+) and primed (CD4+CD45RO+) T cells in peripheral blood mononuclear cells (PBMC) from healthy controls (A), PBMC from patients with rheumatoid arthritis (RA) (B), and synovial fluid mononuclear cells (SFMC) from patients with RA (C). The corresponding profiles for CD45RB are shown for cells within the positive population in each case. CD45RA+ cells are all CD45RBbrigh', but CD45RO' cells show a wide range of expression of CD45RB. The expression of CD45RB and CD45RO is reciprocal within the primed population in control PBMC (D), RA PBMC (E), RA SFMC (F), reactive arthritis PBMC (G), and reactive arthritis SFMC (H). In RA SF, cells are almost exclusively CD45RBd""/CD45RObriBh', but in reactive arthritis SF cells, a large proportion are CD45RBbnBh'.

Expression of CD45RB by CD4+ T cells in the blood and SF of patients with RA. No differences were found between the CD45RB-defined populations of CD4+ T cells in the peripheral blood of patients with RA, patients with reactive arthritis, or healthy con- trols (Figures 1 and 2). In SF from patients with RA, almost all CD4+ T cells were CD45RObngh' and CD45RBdU" (Figure 1). The difference between CD45RBd"" populations in blood and SF was highly significant (P < 0.001). Clearly, part of this can be accounted for by the difference in the proportion of

primed cells at these sites, but when the primed fractions in the peripheral blood and the SF of RA patients were compared, the latter compartment showed a considerably elevated level of CD45RBd"" cells (Figures 1 and 2). In peripheral blood from healthy controls or patients with RA, non-CD4 cells were predominantly CD45RBbngh'; however, in SF, there was a significantly lower proportion of CD45RBbright cells within the non-CD4 cell population (mean -+ SD 76 2 10% in blood, versus 38 % 21% in SF; P < 0.001).

606

Control PBMC

Rheumatoid arthritis PBMC

Rheumatoid arthritis SFMC

Reactive arthritis PBMC

Reactive arthritis SFMC

I

F- **

20 40 6 0 8 0 100

Proportion of CD45RBbright cells

Figure 2. Proportion of CD45RBb*lgh' T cells in the total CD4+ fraction (0) and the CJM+CD45RO+ primed fraction (=) of peripheral blood from 18 healthy controls compared with peripheral blood and SF from 10 patients with RA and 5 patients with reactive arthritis. SF T cells from patients with RA or reactive arthritis showed a considerably elevated proportion of the CD4SRBd"" subset within the total CD4+ fraction, compared with autologous peripheral blood (P < 0.001 by Mann-Whitney U test) (**). How- ever, patients with reactive arthritis had significantly more SF CD45RBbnSh' cells than did patients with RA (P < 0.02 by Mann- Whitney U test) (#). There were no apparent differences from control in the peripheral blood profile of either patient group. RA SF represents an almost exclusively primed population, almost all of which is CD45RBdU", while the primed fraction of CD4+ cells in SF from patients with reactive arthritis differs little from that fraction in peripheral blood. Values are the mean and SD. See Figure 1 for definitions.

CD45 isoform expression in patients with reac- tive arthritis. The 5 patients with acute reactive arthri- tis showed similar profiles of peripheral blood CD45 isoform expression to the peripheral blood profiles in healthy controls and patients with RA, but their SF expression was quite distinct (Figures 1D-H and 2). There was higher expression of CD45RA, which is almost absent in RA SF, and, more significantly, the number of CD45RBbright cells within the primed frac- tion was 2-fold greater than in patients with RA ( P < 0.02) (Figure 2). In SF from several of the patients with reactive arthritis, we identified a population of large blast cells that were CD45RBbrigh' but intermediate in

MATTHEWS ET AL

expression for CD45RA and CD45R0, suggesting pri- mary activation of virgin T cells (Figure 1H).

DISCUSSION lsoforms of the leukocyte common antigen

(CD45) have been used for several years to divide T helper cells into virgin and primed subpopulations (2,ll-13). CD4+, CD45RA+ cells produce high levels of IL-2, but no IL-4 or IFNy, while primed CD45RO+ cells produce all 3 lymphokines (14,15).

Combined analysis of 3 isoforms in human CD4+ T cells, i.e., CD45RA, CD45RB, and CD45R0, reveals 3 distinct fractions. CD45RA+ virgin T cells all express high levels of CD45RB. However, the CD45RO+ primed T cells are subdivided into 2 frac- tions expressing high or low levels of CD45RB. Data from our own and other laboratories suggest that a functional distinction also exists between the primed subsets (refs. 9 and 11, and Salmon M, Pilling D, Akbar A: unpublished observations).

Differentiation of T cells following activation is accompanied by a transition from high to low molec- ular weight isoforms of CD45. Loss of CD45RA is rapid, requiring only a single cell cycle; however, CD45RB expression is lost gradually over a number of cycles. Thus, CD45RBbrigh' primed cells reflect an early stage of differentiation. In this study we have shown that SF T cells from patients with established RA show a very high degree of selection of the CD45RBd"" phenotype. In contrast to the almost ex- clusively CD45RBd"" character of RA SF cells, those from patients with acute reactive arthritis contain many CD45RBbrigh' cells. These are frequently large activated blasts, and are also intermediate in their expression of CD45RA and CD45R0, suggesting pri- mary activation of virgin T cells. These findings sug- gest that the CD45 phenotype of RA SF T cells may reflect the chronicity of disease, and may also lead to limitation of inflammatory damage at this site.

Aaron and Paetkau have shown that low-level production of IL-2 by SF T cells appears to be an intrinsic property of these lymphocytes (16); this is compatible with a highly selected population of CD45RBd"" cells. The expression of adhesion mole- cules by CD45RBdU" cells is not markedly different from that by the CD45RO+, CD45RBbrigh' population, and a recent study has shown no selectivity of binding to endothelium for either subset (17). In vitro, the progression from high expression of CD45RB to very low expression takes several weeks of continuous

PRIMED T HELPER CELL SUBSETS IN RA 607

activation (9). There appears to be little evidence fo r selective recruitment of CD45RBd"" cells to the syno- vium, while the primed (CD45RO+) population in peripheral blood contains a substantial proportion of CD45RBbright cells; the almost exclusively CD45RBd"" phenotype of SF cells therefore suggests either in situ proliferation or, perhaps, a quite separate mechanism of phenotype conversion which we cannot reproduce in vitro. Unfortunately it is very difficult to study the CD45RB status of cells in tissue sections since all leukocytes are CD45RB+, but to differing degrees. However it seems probable that many of the reported functional anomalies in SF-derived T cells from pa- tients with RA reflect this highly selected population of cells primed through many cycles of activation.

ACKNOWLEDGMENTS We thank Dr. David Mason for the gift of monoclonal

antibody PD7/26, and Professor Peter Beverley for the gift of monoclonal antibody UCHL-1.

1.

2.

3.

4.

5 .

6.

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