An evaluation of lymphocyte capping in Duchenne muscular dystrophy

Journal of the Neurological Sciences, 1982, 53 : 511-518

51 1

Elsevier Biomedical Press

AN EVALUATION OF LYMPHOCYTE CAPPING IN DUCHENNE MUSCULAR DYSTROPHY

GARTH A. NICHOLSON and JOHN SUGARS

University of Sydney, Clinical Sciences Building, Concord Hospital, Concord. 2139 (Australia) (Received 4 August, 1981) (Accepted 11 August, 1981)

SUMMARY

There have been conflicting reports of lymphocyte capping abnormalities in Duchenne muscular dystrophy (DMD). We have evaluated the original method described by Verrill et al. in a "blind" study of 24 Duchenne muscular dystrophy boys and paired age-matched control boys. We found no differences in capping between the two groups but control boys had decreased capping compared to a group of normal adult males and females. It is concluded that the initial reports of decreased capping in DMD may have been due to differences in age between the test and control groups.

INTRODUCTION

Much recent research in Duchenne muscular dystrophy (DMD) has concentrated on investigation of cell membrane functions, on the assumption that a generalised disorder of cell membranes occurs in this disease (Rowland 1980). Although many membrane abnormalities have been described, there is no firm evidence that the primary molecular defect involves the cell membrane. Not one of the membrane biochemical alterations has proved superior to existing methods for DMD carrier detection. Lymphocyte capping depends on both membrane and intracellular functions. The molecular basis of capping is not well understood but may involve actin, microfilaments and microtubules. Capping could be affected in DMD lymphocytes since This work was supported by a grant from the Muscular Dystrophy Association of the U.S.A. Address for reprints: Dr. G.A. Nicholson, Department of Medicine, Clinical Sciences Building, Concord Hospital, Concord. 2139, Australia. 0022-510X/81/0000-0000/$02.75 © Elsevier Biomedical Press

512 the molecular events in capping may share features with both skeletal muscle contraction and with membrane function. In 1977 Verill et al. reported decreased capping in lymphocytes from both DMD boys and female carriers of the gene and also in Becker, limb-girdle, facioscapulohumeral and some congenital muscular dystrophies. Subsequently, this work was expanded by the same group (Pickard et al. 1978). Similar findings for DMD patients and carriers were reported by Ho et al. (1980). Bader (1979) also reported decreased capping in limb-girdle dystrophy patients and in unaffected parents and siblings. Other authors have not been able to repeat these results (Stern et al. 1979; Hauser et al. 1979a,b). In addition, no alteration in lymphocyte capping was found in the Bar Harbour (129 dy/dy) dystrophic mouse (Hauser et al. 1979b). The inability of subsequent investigators to reproduce the original results could be due to differences in methodology. Most of these studies did not use the same experimental conditions as in the original report. The incubation buffer (Seligmann's balanced salt solution) used in the original studies by Verrill and Pickard contains no calcium and presumably could affect the result of capping in DMD, a disorder where there are known abnormalities of intracellular calcium metabolism (Bodensteiner et al. 1978). Some studies used only small numbers of patients and some omitted matched controls. Different fluorescein labelled antiimmunoglobulins can produce staining variations (Sybert et al. 1979), and the results of counting capped cells may vary according to the selection criteria for classifying cells as capped, patched, clustered or uniform. Allocation of staining categories is a subjective assessment and perhaps could be influenced by knowledge of the likely outcome. For this reason capping is best assessed in a "blind" fashion where the observer does not know the origin of the sample under study. In some of the studies mentioned above it is not possible to determine whether the study was performed "blind". In a recent paper,Horenstein and Emery (1980) found decreased capping in DMD lymphocytes but these authors used a different capping method and nonage-matched controls. In order to resolve these conflicting reports we have undertaken a "blind" evaluation of lymphocyte capping using the original method and reagents as described by Verrill and Pickard. We have restricted our study to DMD boys and age- and sex-matched controls. MATERIALSAND METHODS DMD patients were referred from the Prince of Wales Children's Hospital, Muscular Diseases Clinic. The diagnosis was established on clinical grounds, serum creatine kinase estimation and muscle biopsy. Age- and sex-matched non-related control boys included friends of DMD families and volunteers from schools. Subjects with recent illness were excluded. Blood samples were obtained simultaneously from normal boys and DMD patients. Samples were coded so that the origin of the cells was not known to the observer until all results were analysed.

513 Fifteen millilitres of whole blood were collected in plastic tubes with 150 units ofheparin. Cell separations were carried out within 1 h and generally within 10 min of collection. The method used was exactly as described by Pickard et al. (1978). Lymphocyte yields were determined from whole blood differential white cell counts using Max-Griinwald and Giemsa stains. Seligmann's balanced salt solution was made up according to the standard formula (H. Verrill, personal communication). Ficoll-Paque was obtained from Pharmacia Fine Chemicals. Fluorescein-labelled polyvalent anti-human immunoglobulin was obtained from Meloy Springfield, VA. Fluorescein-labelled Con A was obtained from the Sigma Chemical Company. RESULTS

Incubation conditions The effect of varying incubation times up to 45 min at 37 °C was determined, since too long incubation could potentially result in endocytosis of caps. Capping increased with time approximately linearly. Maximum labelling occurred at 1/10 dilution of fluorescein-labelled polyvalent anti-human immunoglobulin.

Lymphocyte yields Yields of lymphocytes were determined from D M D and control blood samples (Fig. 1). No differences were found (P > 0.4).

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Fig. 1. A : Lymphocyte yields from D M D patients and normal subjects. B: Percentages of cells labelled with fluorescein-conjugated anti-human immunoglobulin. Dotted lines indicate mean values.

514

Percentage labelling Percentages of fluorescein-labelled anti-immunoglobulin-stained cells were determined in D M D and control lymphocytes (Fig. 1). No differences were found (P > 0.05).

Lymphocyte capping in DMD boys and controls Lymphocyte capping was tested in samples from 24 D M D patients and ageand sex-matched controls. No difference was found in lymphocyte capping using either anti-human immunoglobulin (P > 0.02, Fig. 2, Table 1) or fluorescein-labelled 80

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Fig. 2. Lymphocyte capping in DMD patients and normal subjects with fluorescein-labelled anti-human immunoglobulin. Dotted lines indicate mean results.

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Fig. 3. Lymphocyte capping results in DMD patients and normal subjects using fluorescein-conjugated concanavalin A. Dotted lines indicate mean values.

515 Con A (P >0.5, Fig. 3, Table 2), whereas differences were noted between adult females and normal boys (Table 3) using anti-human immunoglobulin. TABLE 1 L Y M P H O C Y T E C A P P I N G W I T H POLYVALENT A N T I - H U M A N I M M U N O G L O B U L I N

Capping mean 5: SD

DMD N = 24

36.8* 5:16.6

Controls N = 24

31.8 5:13.4

* Not significantly different from controls (P > 0.2).

TABLE 2 L Y M P H O C Y T E CAPPING W I T H C O N A

~o Capping (mean + SD) DMD N=13

17.4" + 5.9

Controls N = 14

18.6 + 5.4

* Not significantly different from controls (P >0.5).

TABLE 3 L Y M P H O C Y T E C A P P I N G W I T H A N T I - H U M A N I M M U N O G L O B U L I N : N O R M A L SUBJECTS Adult females were aged from 19 to 49 years, mean 29 years, adult males were aged from 26 to 64 years, mean 41 years, boys ranged from 4 to 16 years old; with a mean 9 years.

Capping (mean + SD) Adult females N = 20

44.0* __. 9.0

Adult males N=15

38.6 + 5.4

Boys N = 24

31.8 + 13.4

* Capping results for adult females were significantly different from adult males (P < 0.05), boys (P < 0.01).

516 DISCUSSION Decreased lymphocyte capping has been shown in chronic lymphatic leukaemia (Cohen et al. 1975), autoimmune thromobytopenic purpura (Quagliata et al. 1979), autosomal dominant cerebrovascular amyloidosis (Stefanson et al. 1979), and Huntington's chorea (Noronha et al. 1979). Experience in DMD has been variable. Four groups report decreased capping (Verrill et al. 1977; Bader 1979; Ho et al. 1980; Horenstein et al. 1980). Five other groups (including this report) have not found decreased capping in DMD (Gershwin et al. 1979; Hauser et al. 1979b; Stern et al. 1979; Sybert et al. 1979). Most investigators have used slightly different experimental conditions to those described by the original authors. However, using identical conditions and reagents we did not find any differences in capping between DMD subjects and age- and sex-matched controls. Three theoretical considerations cast doubt on lymphocyte capping as a specific indicator of muscle disease. First, the biochemical mechanism is poorly understood and has not been directly related to muscle metabolism. Second, if the reports of decreased capping in the other diseases cited above are correct, capping must be regarded as a non-specific phenomenon. Third, 3 of the groups reporting decreased capping in DMD also found a quantitatively similar abnormality in carriers. This latter finding is difficult to explain as some mothers may be genetically normal having had a DMD son as a result of a recent mutation and at least a proportion of mothers should be phenotypically normal as a result of non-random inactivation of the DMD X chromosome. Therefore, some mothers would be expected to have normal capping. Methodological differences could also explain the variable results. In particular, if the incubation time with anti-immunoglobulin is not optimal, endocytosis of the caps may occur and any differences between disease and control groups may be lost. Hauser et al. (1979a) stated that capping occurred at 5 min. However, our results showed no decrease in capping over a 45-min period. We therefore elected to use a 30-min incubation as described by Verrill et al. in order to preserve uniformity of method. Other authors, Ho et al. (1980) and Bader (1979), also used a 30-min incubation period. Another potential problem with a quantitative capping assay is the presence of clumped cells. Clumping could result from interaction of specific cell surface determinants on a subgroup of lymphocytes and could possibly deplete the assay of a lymphocyte population responsible for the DMD capping defect. If clumping depends on incubation conditions, variations in incubation conditions could also alter the subgroup of lymphocytes being quantified. Some of the reported decreases in DMD capping could be due to variations in capping related to the age and sex of the control subjects. Indeed, Haber did find decreased capping in parents and siblings, suggesting incorrect normal values. In this study significant differences in capping were found between normal boys and adult females suggesting the presence of age- and/or sex-related changes in capping. Age differences in lymphocyte capping responses have not been reported,

517 but since many immunological reactions do alter with age, we have elected to use age-matched controls. Most studies of the effect of diseases on lymphocyte capping have not employed age- and sex-matched controls. If an increase in capping does occur with increasing age of the subject then this effect could explain all reports of differences between D M D patients and controls. Since we have not found any disease related differences in capping in D M D we did not study D M D carriers as they would be unlikely to show any abnormality. In conclusion, until the mechanism and sources of variability of lymphocyte capping are better understood, lymphocyte capping does not constitute a reliable method for Duchenne musclalar dystrophy carrier detection. ACKNOWLEDGEMENTS

The assistance of patients and relatives attending the Prince of Wales Muscle Diseases Clinic, Dr. Graeme Morgan, Director of the Clinic, Sister Kroon of the Child Minding Centre, Concord Hospital and the parents and children of Concord West Public School is gratefully acknowledged. REFERENCES Bader, P. I. (1979) Lymphocyte capping in carriers of Duchenne muscular dystrophy, N. Engl. J. Med., 301 : 725 (Letter to the Editor). Bodensteiner, J.P. and A.G. Engel (1978) Intracellular calcium accumulation in Duchenne dystrophy and other myopathies - - A study of 567,000 muscle fibres in 114 biopsies, Neurology (Minneap.), 28 : 439446. Cohen, H.J. and B.B. Gilbertsen (1975) Human lymphocyte surface immunoglobulin c a p p i n g Normal characteristics and anomalous behaviour of chronic lymphocytic leukaemic lymphocytes, J. clin. lnvest., 55: 84-93. Gershwin, M. E., R. G. Taylor, W. M. Fowler and B. Finlayson (1979) Failure to demonstrate abnormal lymphocyte capping in humans, mice and hamsters with muscular dystrophy, Hum. Genet., 53: 113-114. Hauser, S. L., H. Weiner, K. Ault and E. Unanue (1979a) Lymphocyte capping in Duchenne muscular dystrophy, N. Engl. J. Med., 300:861 (Letter to the Editor). Hauser, S.L., H.L. Weiner, M.D. Bresnan and K.A. Ault (1979b) Lymphocyte capping in muscular dystrophy, Neurology (Minneap.), 29: 1419-1421. Ho, A.D., B. Reitter, S. Stojakowits, W. Fiehn and J. Weisser (1980) Capping of lymphocytes for carrier detection in Duchenne muscular dystrophy - - Technical problems and a review of the literature, Europ. J. Pediat., 134: 211-216. Horenstein, A.L. and A.E.H. Emery (1980) Human lymphocyte capping in Duchenne muscular dystrophy, Neurology (Minneap.), 30 : 1330-1332. Noronha, A.B.C., R.P. Roos, J.P. Antel and B.G.W. Arnason (1979) Huntington's disease - Abnormality of lymphocyte capping, Ann. Neurol., 6 : 447~t50. Pickard, N. A., H. D. Gruemer, H. L. Verrill, E. R. Isaacs, M. Robinow, W. E. Nance, E.C. Myers and B. S. Goldsmith (1978) Systemic membrane defect in the proximal muscular dystrophies, N. Engl. J. Med., 229: 841-846. Quagliata, F. and K. Karpatkin (1979) Impaired lymphocyte transformation and capping in autoimmune thrombocytopenic purpura, Blood, 53: 341-349. Rowland, L.P. (1980) Biochemistry of muscle membranes in Duchenne muscular dystrophy, Muscle and Nerve, 3 : 3-20. Stefanson, K., J.P. Antel, J. Oger, J. Burns, B.C. Avertano, M.B. Norohna, R.P. Roos, B.G.W. Arnason and G. Gudmundsson (1979) Autosomal dominant cerebrovascular amyloidosis - Properties of peripheral blood lymphocytes, Ann. Neurol., 7: 436~,40.

5~8 Stern, C.M.M., M.C. Kahan and V. Dubowitz (1979) Lymphocyte capping in Duchenne muscular dystrophy, Lancet, i: 1300. Sybert, V.P., K.N. Setran and M.E. Kadin (1979) Lymphocyte capping in carriers of Duchenne muscular dystrophy, N. Engl. J. Med., 301:724-725 (Letter to the Editor). Verrill, H. L., N. A. Pickard and H. D. Gruemer (1977) Diminished cap formation in lymphocytes from patients and carriers of Duchenne muscular dystrophy, Clin. Chem., 23: 2341-2343.