Absence of a correlation between T lymphocyte subsets and clinical activity in relapsing-remitting multiple sclerosis

Journal of Neuroimmunology, 22 (1989) 41-45

41

Elsevier JNI 00723

Absence of a correlation between T lymphocyte subsets and clinical activity in relapsing-remitting multiple sclerosis R. C a p r a 1, F. Mattioli

1, L.A. Vignolo 1, M.

Bettinzioli

2, R.

C a t t a n e o 2 a n d F. Franceschini 2

l Clinica Neurologica, and 2 Istituto di Immunologia Clinica, University of Brescia, Brescia, Italy

(Received 25 February 1988) (Revised, received3 August 1988) (Accepted 3 August 1988)

Key words: Multiplesclerosis, relapsing-remitting;Lymphocytesubpopulation

Summary Cytofluorographic analysis of CD3 ÷, Tac ÷, HLA-DR ÷ peripheral blood lymphocytes and C D 4 / C D 8 ratio was performed monthly, over a 10-month period, in a group of 16 patients with multiple sclerosis (MS). No correlation was found between clinical relapses and fuctuations in the lymphocyte subsets, although patients who were in remission throughout the study showed a number of CD3 ÷ cells and a C D 4 / C D 8 ratio significantly lower than those in normal controls. We concluded that changes in peripheral blood lymphocyte subsets are not related to the disease process and their measurement is not helpful in monitoring the illness.

Introduction

Although several immunological abnormalities are described in multiple sclerosis (MS) (Link and Tibbling, 1977; Bach et al., 1980; Mehta et al., 1982; Antel et al., 1984; Waksman, 1985; Hauser et al., 1986), their significance is unknown. It has been suggested that MS is a T cell-mediated autoimmune disease (Waksman, 1985) and an increased ratio between peripheral blood CD4 + T lymphocytes (the helper-inducer lymphocytes) and CD8 ÷ T lymphocytes (the suppressor-cytotoxic) has been reported by some authors in relapses

Address for correspondence: R. Capra, Clinica Neurologica, University of Brescia, P.zza Spedali Civili 1, 25125 Brescia, Italy.

(Reinherz et al., 1980; Cashman et al., 1982; Hauser et al., 1983; Caputo et al., 1987), but was not confirmed by others (Sandberg-Wollheim, 1983; Steck and De Flaugergues, 1984). A correlation between increased levels of activated T cells in peripheral blood and clinical progression of the disease has also been recently reported (Selmay et al., 1986). Serial investigations have been performed in a limited number of these studies (Hauser et al., 1983; Thompson et al., 1986; Caputo et al., 1987; Gonsette et al., 1987; Hutchinson et al., 1987) and no straightforward relationship between changes in T cell percentage and worsening of the disease has been identified (Thompson et al., 1986; Gonsette et al., 1987; Hutchinson et al., 1987). In order to see if there was such a relationship between fluctuations in T cell levels and the clini-

0165-5728/89/$03.50 © 1989 Elsevier SciencePublishers B.V.(Biomedical Division)

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cal course in MS, we studied 16 well-characterized patients with relapsing-remitting disease, over a 10-month period.

Patients and methods

Patients The 16 patients were diagnosed as having a clinically definite MS of the relapsing-remitting type by standard criteria (Poser et al., 1983). They were selected on the basis of the clinical course of the illness (relapse or remission) and of their consent for a monthly clinical assessment including the EDSS (Expanded Disability Status Scale) and venopuncture. The monthly assessment was chosen because of the report by Compston (1983) that abnormalities in certain lymphocyte subsets returned to normal within approximately 1 month of the onset of new symptoms. The patients were two men and 14 women. At the beginning of the study, their ages were between 19 and 42 years (mean = 29.6 years) and their duration of illness was between 1 and 14 years (mean = 5.2 years). The EDSS grade (Kurtzke, 1983) was between 0 and 3 (mean = 1.7). The patients' characteristics at the beginning of the study are summarized in Table 1. 20 sex- and age-matched healthy subjects were used as controls, so that normative values for each lymphocyte subset were available. Methods Peripheral blood samples were collected at monthly intervals over a 10-month period. Venopuncture was consistently performed between 8.00 and 8.30 a.m., in order to avoid any circadian variation in the lymphocyte subpopulations (Ritchie et al., 1983).

We were unable to evaluate subsets' changes during clinical worsening without therapy, for ethical reasons: relapses were treated with steroids. Hauser et al. (1983), however, have reported that fluctuations in the C D 4 + / C D 8 + ratio are not induced by steroid therapy. Subset analysis. Cytofluorographic analysis of peripheral blood lymphocytes was performed using fluorescein-labelled monoclonal antibodies, antiCD3, anti-CD4 and anti-CD8 (Ortho, Raritan, N J), by flow cytometry (Spectrum III, Ortho). Briefly, 100 /~1 of heparinized venous blood was incubated with the monoclonal antibody at the appropriate concentration for 30 min at 4°C. After two washes with phosphate-buffered saline (PBS) the cells were resuspended in 2 ml of lysing reagent (Ortho) and examined by flow cytometry. Staining with monoclonal antibodies anti-CD25 (Coulter Clone, Hialeah, FL) and anti-HLA-DR (B33.1, gift of Dr. Bice Perussia, Wistar Institute, Philadelphia) was performed by indirect immunofluorescence using a F(ab')2-FITC-conjugated goat anti-mouse Ig, as a second step and examined by flow cytometry.

Statistics We compared the means of the lymphocyte subpopulations in patient remissions and relapses, using Student's t test. In addition, non-parametric statistics were used: the differences between relapses and remissions were compared by the Mann-Whitney U test and the correlation between clinical state and lymphocyte subset quantities in each patient was computed by the Spearman's rank test (Siegel, 1956). The latter was used in only 11 of the 16 patients because the remaining five patients had no relapses during the observation period.

TABLE 1 PATIENTS' CHARACTERISTICS AT T H E ONSET OF T H E S T U D Y

Number

Sex

16

M 2

Age (years) F 14

Mean 29.6

Range 19-42

Duration of the illness (years) Mean Range 5.2 1-14

Disability (EDSS grade) Mean Range 1.7 0-3

43 TABLE 3

Results A total of 138 peripheral b l o o d subset e x a m i n a tions were carried out d u r i n g the 1 0 - m o n t h period, 111 o n patients i n a remission phase a n d 27 d u r i n g a clinical relapse. Five of the 16 patients showed n o change in their clinical state d u r i n g the study period. Interestingly, the relapses all occurred a r o u n d 4 m o n t h s : F e b r u a r y 1987 (n = 6), M a r c h 1987 ( n = 5), April 1987 ( n = 4) a n d M a y 1987 ( n = 4 ) . T h e m e a n values of C D 3 + T lymphocytes ( c e l l s / m l ) were increased i n patients d u r i n g a relapse, c o m p a r e d to a remission or to the results i n n o r m a l healthy controls. Similarly, the m e a n values of the CD3 + T lymphocytes were decreased i n remission, c o m p a r e d to healthy controls b u t the differences were n o t significant b y either Student's t test or the M a n n - W h i t n e y U test. The C D 4 / C D 8 ratio was increased in relapsing phases c o m p a r e d to the r e m i t t i n g phase but, again, the difference was not statistically signific a n t (Table 2). The m e a n values of Tac + a n d H L A - D R + cell percentages i n relapsing a n d r e m i t t i n g phases fell within the n o r m a l range; the m e a n values i n relapse were slightly, b u t n o t significantly, higher t h a n in remission (Table 2). W e looked for a statistical correlation b e t w e e n the clinical phase of MS (relapse or remission phase) a n d single l y m p h o c y t e subset values in each patient, using S p e a r m a n ' s r a n k test (see T a ble 3). N o n e of the correlations were statistically significant: CD3 + lymphocytes a n d clinical phase showed almost n o correlation in four patients

RANK SPEARMAN'S r VALUES FOR CD3 + CELLS/ml, CD4/CD8 RATIO, HLA-DR + AND Tac + CELLS PERCENTAGES IN 11 PATIENTS WHO SUFFERED A RELAPSE None of these values are statistically significant. Patients

CD3 +'

CD4/CD8

HLA-DR ÷

Tac ÷

A.A.M. B.L. B.M. R.A. C.A. C.M.G. G.A. M.T. M.O. P.P.

0 0.158 0 0.503 0.109 0.069 0.410 0.207 0.522 0.082

0.348 -0.503 0.394 -0.275 0.409 -0.243 -0.410 0.287 0.522 0.082

0 0.474 0.507 0 -0.354 -0.313 0 0 -0.261 0.412

0.058 -0.398 0.507 0.449 -0.218 -0.375 -0.410 0.377 0.166 0.042

( r = 0.089), a positive correlation i n six patients ( r b e t w e e n 0.125 a n d 0.522) a n d a negative o n e i n a n o t h e r p a t i e n t ( r = - 0 . 4 1 0 7 ) . The C D 4 / C D 8 ratio correlation was negative i n five cases (range from r = - 0 . 0 8 2 to r = - 0 . 5 0 3 ) a n d positive i n six (range from r = 0.287 to r = 0.522). The values for H L A - D R + cells (percentage) a n d the clinical phase were i n d e p e n d e n t i n four patients ( r = 0), positive i n a n o t h e r four (range from r = 0.1031 to r = 0.5070) a n d negative i n three (range f r o m r = - 0 . 2 6 1 1 to r = - 0 . 3 5 4 6 ) . F o r Tac + ceils (percentage) correlation was positive i n four p a t i e n t s ( r from 0.166 to 0.507), negative i n four (range from r = - 0 . 2 1 8 2 to r =

TABLE 2 VALUES FOR CD3 ÷ CELLS/ml, CD4/CD8 RATIO, HLA-DR + AND Tac + CELLS PERCENTAGES IN PERIPHERAL BLOOD OF MS PATIENTS WITH RELAPSE OR REMISSION AND IN HEALTHY SUBJECTS Student's t test for CD3 + = NS, for CD4/CD8, for HLA-DR + = NS, for Tac + = NS. Mann-Whitney U test for CD3 = NS, for CD4/CD8 = NS, for HLA-DR ÷ = NS, for Tac ÷ = NS. Patients tested in:

Number of tests

CD3 ÷ (cells/ml) Mean SD

CD4/CD8 Mean SD

Period of relapse Period of remission Healthy subjects

111

1858.7

983.7

2.06

0.89

9.36

27

1484.2

565.7

1.70

0.65

1680

120.0

1.80

0.40

150

HLA-DR+(%) Mean SD

Tac+(%) Mean

SD

5.39

0.91

0.88

9.85

5.09

1.23

1.38

12.05

6.02

2.90

2.70

44 -0.4107) and no correlation was found in three patients (r = 0-0.058). It should be stressed that even the highest correlations failed to reach statistical significance. As shown in Table 3, the values for CD3 + cells/ml, Tac +, H L A - D R + lymphocytes percentage and C D 4 / C D 8 ratio in both relapse and remission show large standard deviations, when compared to the means, so that an overall statistically significant difference can hardly be found. In order to identify any more homogeneous clusters of patients, with the same trend, we divided our population, on the basis of the clinical course, into two main groups: patients in continual remission (five patients) and patients with relapses (11 patients). The former group showed mean values and standard deviations (SD) (CD3 + mean = 1475.05 cells/ml and SD = 472; C D 4 / C D 8 mean = 1.40 and SD = 0.297; Tac + mean = 1.35% and SD = 1.26; H L A - D R + mean = 9.9% and SD = 4.11) very similar to the ones found in remission phases as seen in Table 2, and always lower than those in healthy subjects: the latter group was then divided, according to results, into four further subgroups. In each of them the values for H L A - D R + and Tac + cell percentages showed wide fluctuations, always independent of the clinical state of the patient. With regard to the values for CD3 + (cells/ml) and C D 4 / C D 8 ratio, in the first subgroup (three patients), they varied independently of any relapses, and were always within the healthy subjects' range. In the second group (four patients) values of both CD3 + and C D 4 / C D 8 rose above the usual, in correspondence to a relapse (one of these patients usually had C D 4 / C D 8 ratios higher than normal) and in the third one (three patients) only CD3 + cells were higher than usual during relapses. The last group (one patient) showed a clinically related fluctuation in his C D 4 / C D 8 ratio.

Discussion

Many authors have reported an increased C D 4 / C D 8 ratio with decreased percentages of CD8 + T lymphocytes in the peripheral blood or cerebrospinal fluid of patients with MS undergoing an acute exacerbation (Bach et al., 1980; Rein-

herz et al., 1980; Cashman et al., 1982; Hauser et al., 1983; Caputo et al., 1987). Others have disagreed, failing to find any significant correlation between changes in T cell subsets'and the clinical course (Thompson et al., 1986; Gonsette et al., 1987; Hutchinson et al., 1987). Selmay et al. (1986) described significantly higher percentages of Tac + cells in the peripheral blood of patients with a relapse, compared to those undergoing remission. The majority of authors, however, did not carry out longitudinal surveys. The data obtained from the 10 month follow-up on 16 patients with MS reported here, though revealing certain trends in the number of CD3 + ceils and in the C D 4 / C D 8 ratio, show no correlation between lymphocyte subset fluctuations and the course. Within the group of patients who had clinical relapses (11) seven showea increased CD3 + lymphocyte number near the relapse and five of these had a higher C D 4 / C D 8 ratio in relapse than in remission. The lymphocytopenia induced by steroid therapy (given to relapsing patients) may have played a role in increasing the difference between the number of CD3 + cells in the patients during relapses and in remissions. No correlation was found in remaining patients. On the other hand, the five patients in remission throughout the period did have a correlation between their CD3 + lymphocyte values, C D 4 / C D 8 ratios and clinical state. One can state, therefore, that continual decreased numbers of CD3 + cells and a low C D 4 / C D 8 ratio, characterize a more benign disease. On the other hand, H L A - D R + and Tac + cells percentages (although the antigens are considered to be markers of activation and so the number of such cells might increase in a relapse) showed large fluctuations, independent of the clinical course. Several other factors (e.g., infections, therapies, stresses etc.) may influence the number of activated cells, of CD3 + cells and C D 4 / C D 8 ratio, this making estimation of the lymphocyte phenotype a crude index of immunoregulation in most autoimmune diseases. The usefulness of studies on peripheral blood lymphocytes in diseases, such as MS, which affect the central nervous system, is open to debate; the lack of knowledge about the pathogenetic reac-

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tions involved in this disease indicates that caution must be exercized before using the lymphocyte subset determinations in clinical management. Clinical examination alone cannot detect each episode of progressive demyelination in the central nervous system since clinically silent plaques can appear and disappear (Oger et al., 1987). It may be that serial magnetic resonance imaging assessments will turn out to be of the most use to the clinician (De Dominicis et al., 1987; Lee and Rao, 1987).

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