- Email: [email protected]
HAIRY CELLS
976 Serum samples from donors were initially screened at a 1/8 dilution against test cells only. Approximately 1 in 200
false-positive screen test, but these were readily as such by confirmatory tests.22 North London Blood Transfusion J. BARBARA Centre, J. V. DENNING Deansbrook Road,
gave
a
classified
Edgware, London HA8 Department of Virology, Middlesex Hospital, London W1P 7LD.
9BD.
T. E. CLEGHORN. D. S. DANE MOYA BRIGGS.
INTESTINAL LYMPHOCYTES IN CROHN’S DISEASE editorial SIR,-Your (Jan. 11, p. 80) on Crohn’s disease
(c.D.) emphasised the enigmatic nature of this disease. The frequent absence of both tuberculin sensitivity and skin reactivity to 2,4 dinitrochlorobenzene (D.N.C.B.) in patients with c.D. has raised the question of depressed cellular immune responsiveness in C.D.4,5 Depressed in-vitro functions of peripheral blood lymphocytes from patients with C.D. have been described. 6,7 Thus, the attempt was warranted to isolate lymphocytes directly from the affected intestinal tissue after operation and to examine their immunological properties. As shown previously,8.9 it is possible to obtain viable lymphocytes from freshly operated human gut and appendices. Using this procedure, we isolated viable lymphocytes from the lamina propria of resected small-bowel specimens of 7 patients with a histologically verified diagnosis of c.D. Small-intestine specimens from 5 patients operated on for non-inflammatory conditions served as controls. Any steroid treatment of patients with c.D. had been discontinued for more than 7 days before operation. The viability of isolated lymphocytes was >90%, as judged by the trypanblue exclusion test. As a functional test the reactivity to phytohaemagglutinin (P.H.A.) in short-term tissue cultures was assessed, using the optimally stimulating dose of P.H.A. Rosette formation of lymphocytes was allowed to occur under conditions described by Jondal et apo Lymphocyte suspensions isolated from normal small intestine contained a mean of 35-4% rosette-forming lymphocytes, whereas 4. 5. 6. 7.
Jones Williams, W. Gut, 1965, 6, 503. Jones, J. V., Housley, J., Ashurst, P. M., et al. ibid. 1969, 10, 52. Walker, J. G., Greaves, M. F. ibid. p. 414. Brown, S. M., Taub, R. N., Present, D. H. et al. Lancet, 1970, i,
8.
Breucha, G., Riethmüller, G., Rieber, E. P. Klin. Wschr. (in the press). Breucha, G., Riethmüller, G., Saal, J. G. Z. Immun.-Forsch. 1974, 147, 333. Jondal, M., Holm, G., Wigzell, H. J. exp. Med. 1972, 136, 207.
lymphocytes isolated from histopathologically verified C.D. was in the range of 49-5%-79-5% (see accompanying table). The lymphocytes isolated from the patient group showed a suppressed response to P.H.A. with stimulation indices ranging from 10-5 to 20-7 compared with lymphocytes from histologically normal small-bowel specimens, for which stimulation indices ranged from 67-0 to 225 3. Since P.H.A. predominantly stimulates T cells, it is suggested that gut T lymphocytes from patients with c.D. are deficient in blastogenic response to this T-cell mitogen.
the corresponding figure for small-bowel specimens with
The reasons for this defect are unknown. In various viral infections a depression of delayed-type hypersensivity has been observed, and also the response of lymphocytes to P.H.A. is impaired by certain viruses in vitr0.11 It would be premature to attribute the decreased reactivity of gut lymphocytes from patients with Crohn’s disease to the presence of a virus. Further studies are needed to describe the immunological activities of lymphocytes present in the gut. A particular point of interest is the antigenic specificities of the lymphocytes isolated from the granulomatous lesion itself. Chirurgische Klinik der Universität Tübingen, D 7400 Tübingen, West Germany.
G. BREUCHA G. RIETHMÜLLER.
HAIRY CELLS
SiR,—After demonstrating, by scanning electron micro(S.E.M.), hairy lymphocytes in the peripheral blood and smooth lymphocytes in a lymph-node from a patient with chronic lymphocytic leukemia (C.L.L.), Dr Brynes and his colleagues (March 22, p. 687) postulated an inherent difference in the surface morphology of circulating versus non-circulating malignant lymphocytes.12 Earlier, Dr Catovsky and his associates (Feb. 22, p. 462) postulated different surface morphology for hairy cells of leuksmic reticuloendotheliosis in the blood compared with the same cells in the spleen. On the basis of our study of the blood from 3 patients with C.L.L.,12 we suggest that the findings of these investigators result from the effects of preparatory procedures. Most normal or C.L.L. lymphocytes show few microvilli, on S.E.M. or phase, microscopy, when fixed promptly upon removal from circulation by pouring the whole blood directly into glutaraldehyde fixative.12.13 B-lymphocytes scope
1112.
9. 10.
11. Wheelock, E. F., Toy, St. T. Advanc. Immun. 1973, 16, 124. 12. Katayama, I., Valacer, D. J., Yang, J. P. S., Schneider, G. B., Pechet, L. Unpublished. 13. Michaelis, T. W., Larrimer, N. R., Metz, E. N., Balcerzak, S. P., Blood, 1971, 37, 23.
ROSETTE-FORMING CELLS AND P.H.A. RESPONSE IN LYMPHOCYTE SUSPENSIONS ISOLATED FROM HUMAN SMALL-BOWEL TISSUE IN DISEASE
CROHN’S
977 of the bursa of Fabricius also showed smooth surfaces when fixed in situ.14 In contrast, when fixed after preparatory procedures such asFicoll’-’ Hypaque’ separation, commonly used for s.E.M., the majority of C.L.L. lymphocytes show numerous microvilli.12 Therefore although not described in Dr Brynes’ letter, we suspect the preparation of the blood included either dextrose or ficoll-hypaque
separation. On the other hand, the photomicrograph from lymph-node showing smooth lymphocytes both in the capillary and in the tissue suggests en-bloc fixation immediately upon removal.
the
University of Massachusetts Medical School, Worcester, Mass. 01605, U.S.A.
I. KATAYAMA LIBERTO PECHET.
NORMARSKI OPTICS AND T AND B MARKERS SIR,- The letter by Sciorra and Eckert 15 prompted"us to correlate lymphocyte surface morphology (" smooth and hairy ") as observed by Normarski interference phase microscopy with markers of T and B cells such as the ability to form rosettes with sheep erythrocytes (T cell) and presence of membrane-bound immunoglobulin (B cell). "
Blood was drawn by venepuncture from 9 healthy volunteer donors and 5 patients with chronic lymphatic leukaemia. The lymphocytes were separated by centrifugation through a ’FicollHypaque’ density gradient 16 and processed and examined within two hours. The cells were stained for membrane-bound immunoglobulin with fluorescent rabbit polyvalent anti-human immunoglobulin as a marker of B cells, and incubated with 1 % sheep erythrocytes to demonstrate E-rosette-forming T cells. Our techniques have been described elsewhere.17 A wet preparation of each lymphocyte suspension was examined at x 1250 using a Leitz Orthoplan microscope with a Normarski interference contrast attachment. Cells were classified as " hairy " if they had prominent microvilli or three or more hairs longer - than at least a quarter the diameter of the cell. Fluorescein-stained cells were identified by means of a Ploem vertical illuminator incorporated into the Normarski unit and equipped with a HB0200 mercury-vapour lamp with 390NM exciter and 510NM barrier filters. Incubation at 37°C for three hours or prolonged contact of the
Holbrook, K. A., Perkins, W. D., Glick, B. J. reticuloendothel. Soc. 1974, 16, 300. 15. Sciorra, L., Eckert, C. Lancet, 1974, ii, 1526. 16. Boyum, A. Scand. J. clin. Lab. Invest. 1968, suppl. 21, p. 97. 17. Ramachandar, K., Douglas, S. D., Siltzbach, L. E., Taub, R. N. Cell. Immun. (in the press).
cell suspension with a glass coverlip did the proportion of hairy and smooth cells.
not
significantly change
The results are summarised in the accompanying table. In the normal donors, the values obtained for smooth lymphocytes were similar to the proportion of cells forming E-rosettes; but the individual values correlated poorly. Similarly, the proportion of hairy lymphocytes did not correlate significantly with immunoglobulin-positive cells. In the c.L.L. patients a high proportion of the cells assayed showed surface immunoglobulin, yet showed a high proportion of smooth cells by the Normarski technique. Moreover, when the cells of 4 normal donors and 3 C.L.L. patients were examined by simultaneous Normarski optics and immunofluorescence, many smooth cells were directly seen to be immunoglobulin-positive. Similar findings have been recorded in c.L.L. lymphocytes examined by immunoelectron microscopy. IS When E-rosettes from either normal or C.L.L. lymphocytes were examined by Normarski optics no hairy cells were seen to form E-rosettes. These observations suggest that there are indeed two populations of lymphocytes (hairy and smooth) that can be identified by Normarski interference contrast microscopy, but they do not correspond with the population of thymus and bone-marrow derived lymphocytes as assayed by other markers. From our data it is reasonable to assume the E-rosette-forming cells are contained within the smooth lymphocyte population. The relationship between the surface topography of lymphocytes observed using this system and that seen using the critical-point drying technique and scanning electron microscopy remains to be clarified. For example, the almost uniform smooth-cell surface appearance in the 5 cases of C.L.L. that we observed contrasts with the observations of Polliak et al.,19 where cells showed numerous surface microvilli. This work was supported in part by U.S.P.H.S. grants CA-14502 and CA-1287 from the National Cancer Institute. Transplantation Immunology Laboratory and Division of Hematology, JOSE M. VILA Mount Sinai School of Medicine, ROBERT N. TAUB. New York, N.Y. 10029, U.S.A.
14.
18. Reyes, F., Lejonc, J. L., Gourdin, M. F., Mannoni, P., Dreyfus, B. J. exp. Med. 1975, 141, 392. 19. Polliak, A., Lampern, N., Clarkson, B. D., DeHarven, E., Bentwich L., Siegel, F. P., Kunkel, H. G. ibid. 1973, 138, 607.
RESULTS WITH NORMARSKI OPTICS AND WITH T AND B MARKERS
I
*
.
r
=
correlation coefficient.
,
false-positive screen test, but these were readily as such by confirmatory tests.22 North London Blood Transfusion J. BARBARA Centre, J. V. DENNING Deansbrook Road,
gave
a
classified
Edgware, London HA8 Department of Virology, Middlesex Hospital, London W1P 7LD.
9BD.
T. E. CLEGHORN. D. S. DANE MOYA BRIGGS.
INTESTINAL LYMPHOCYTES IN CROHN’S DISEASE editorial SIR,-Your (Jan. 11, p. 80) on Crohn’s disease
(c.D.) emphasised the enigmatic nature of this disease. The frequent absence of both tuberculin sensitivity and skin reactivity to 2,4 dinitrochlorobenzene (D.N.C.B.) in patients with c.D. has raised the question of depressed cellular immune responsiveness in C.D.4,5 Depressed in-vitro functions of peripheral blood lymphocytes from patients with C.D. have been described. 6,7 Thus, the attempt was warranted to isolate lymphocytes directly from the affected intestinal tissue after operation and to examine their immunological properties. As shown previously,8.9 it is possible to obtain viable lymphocytes from freshly operated human gut and appendices. Using this procedure, we isolated viable lymphocytes from the lamina propria of resected small-bowel specimens of 7 patients with a histologically verified diagnosis of c.D. Small-intestine specimens from 5 patients operated on for non-inflammatory conditions served as controls. Any steroid treatment of patients with c.D. had been discontinued for more than 7 days before operation. The viability of isolated lymphocytes was >90%, as judged by the trypanblue exclusion test. As a functional test the reactivity to phytohaemagglutinin (P.H.A.) in short-term tissue cultures was assessed, using the optimally stimulating dose of P.H.A. Rosette formation of lymphocytes was allowed to occur under conditions described by Jondal et apo Lymphocyte suspensions isolated from normal small intestine contained a mean of 35-4% rosette-forming lymphocytes, whereas 4. 5. 6. 7.
Jones Williams, W. Gut, 1965, 6, 503. Jones, J. V., Housley, J., Ashurst, P. M., et al. ibid. 1969, 10, 52. Walker, J. G., Greaves, M. F. ibid. p. 414. Brown, S. M., Taub, R. N., Present, D. H. et al. Lancet, 1970, i,
8.
Breucha, G., Riethmüller, G., Rieber, E. P. Klin. Wschr. (in the press). Breucha, G., Riethmüller, G., Saal, J. G. Z. Immun.-Forsch. 1974, 147, 333. Jondal, M., Holm, G., Wigzell, H. J. exp. Med. 1972, 136, 207.
lymphocytes isolated from histopathologically verified C.D. was in the range of 49-5%-79-5% (see accompanying table). The lymphocytes isolated from the patient group showed a suppressed response to P.H.A. with stimulation indices ranging from 10-5 to 20-7 compared with lymphocytes from histologically normal small-bowel specimens, for which stimulation indices ranged from 67-0 to 225 3. Since P.H.A. predominantly stimulates T cells, it is suggested that gut T lymphocytes from patients with c.D. are deficient in blastogenic response to this T-cell mitogen.
the corresponding figure for small-bowel specimens with
The reasons for this defect are unknown. In various viral infections a depression of delayed-type hypersensivity has been observed, and also the response of lymphocytes to P.H.A. is impaired by certain viruses in vitr0.11 It would be premature to attribute the decreased reactivity of gut lymphocytes from patients with Crohn’s disease to the presence of a virus. Further studies are needed to describe the immunological activities of lymphocytes present in the gut. A particular point of interest is the antigenic specificities of the lymphocytes isolated from the granulomatous lesion itself. Chirurgische Klinik der Universität Tübingen, D 7400 Tübingen, West Germany.
G. BREUCHA G. RIETHMÜLLER.
HAIRY CELLS
SiR,—After demonstrating, by scanning electron micro(S.E.M.), hairy lymphocytes in the peripheral blood and smooth lymphocytes in a lymph-node from a patient with chronic lymphocytic leukemia (C.L.L.), Dr Brynes and his colleagues (March 22, p. 687) postulated an inherent difference in the surface morphology of circulating versus non-circulating malignant lymphocytes.12 Earlier, Dr Catovsky and his associates (Feb. 22, p. 462) postulated different surface morphology for hairy cells of leuksmic reticuloendotheliosis in the blood compared with the same cells in the spleen. On the basis of our study of the blood from 3 patients with C.L.L.,12 we suggest that the findings of these investigators result from the effects of preparatory procedures. Most normal or C.L.L. lymphocytes show few microvilli, on S.E.M. or phase, microscopy, when fixed promptly upon removal from circulation by pouring the whole blood directly into glutaraldehyde fixative.12.13 B-lymphocytes scope
1112.
9. 10.
11. Wheelock, E. F., Toy, St. T. Advanc. Immun. 1973, 16, 124. 12. Katayama, I., Valacer, D. J., Yang, J. P. S., Schneider, G. B., Pechet, L. Unpublished. 13. Michaelis, T. W., Larrimer, N. R., Metz, E. N., Balcerzak, S. P., Blood, 1971, 37, 23.
ROSETTE-FORMING CELLS AND P.H.A. RESPONSE IN LYMPHOCYTE SUSPENSIONS ISOLATED FROM HUMAN SMALL-BOWEL TISSUE IN DISEASE
CROHN’S
977 of the bursa of Fabricius also showed smooth surfaces when fixed in situ.14 In contrast, when fixed after preparatory procedures such asFicoll’-’ Hypaque’ separation, commonly used for s.E.M., the majority of C.L.L. lymphocytes show numerous microvilli.12 Therefore although not described in Dr Brynes’ letter, we suspect the preparation of the blood included either dextrose or ficoll-hypaque
separation. On the other hand, the photomicrograph from lymph-node showing smooth lymphocytes both in the capillary and in the tissue suggests en-bloc fixation immediately upon removal.
the
University of Massachusetts Medical School, Worcester, Mass. 01605, U.S.A.
I. KATAYAMA LIBERTO PECHET.
NORMARSKI OPTICS AND T AND B MARKERS SIR,- The letter by Sciorra and Eckert 15 prompted"us to correlate lymphocyte surface morphology (" smooth and hairy ") as observed by Normarski interference phase microscopy with markers of T and B cells such as the ability to form rosettes with sheep erythrocytes (T cell) and presence of membrane-bound immunoglobulin (B cell). "
Blood was drawn by venepuncture from 9 healthy volunteer donors and 5 patients with chronic lymphatic leukaemia. The lymphocytes were separated by centrifugation through a ’FicollHypaque’ density gradient 16 and processed and examined within two hours. The cells were stained for membrane-bound immunoglobulin with fluorescent rabbit polyvalent anti-human immunoglobulin as a marker of B cells, and incubated with 1 % sheep erythrocytes to demonstrate E-rosette-forming T cells. Our techniques have been described elsewhere.17 A wet preparation of each lymphocyte suspension was examined at x 1250 using a Leitz Orthoplan microscope with a Normarski interference contrast attachment. Cells were classified as " hairy " if they had prominent microvilli or three or more hairs longer - than at least a quarter the diameter of the cell. Fluorescein-stained cells were identified by means of a Ploem vertical illuminator incorporated into the Normarski unit and equipped with a HB0200 mercury-vapour lamp with 390NM exciter and 510NM barrier filters. Incubation at 37°C for three hours or prolonged contact of the
Holbrook, K. A., Perkins, W. D., Glick, B. J. reticuloendothel. Soc. 1974, 16, 300. 15. Sciorra, L., Eckert, C. Lancet, 1974, ii, 1526. 16. Boyum, A. Scand. J. clin. Lab. Invest. 1968, suppl. 21, p. 97. 17. Ramachandar, K., Douglas, S. D., Siltzbach, L. E., Taub, R. N. Cell. Immun. (in the press).
cell suspension with a glass coverlip did the proportion of hairy and smooth cells.
not
significantly change
The results are summarised in the accompanying table. In the normal donors, the values obtained for smooth lymphocytes were similar to the proportion of cells forming E-rosettes; but the individual values correlated poorly. Similarly, the proportion of hairy lymphocytes did not correlate significantly with immunoglobulin-positive cells. In the c.L.L. patients a high proportion of the cells assayed showed surface immunoglobulin, yet showed a high proportion of smooth cells by the Normarski technique. Moreover, when the cells of 4 normal donors and 3 C.L.L. patients were examined by simultaneous Normarski optics and immunofluorescence, many smooth cells were directly seen to be immunoglobulin-positive. Similar findings have been recorded in c.L.L. lymphocytes examined by immunoelectron microscopy. IS When E-rosettes from either normal or C.L.L. lymphocytes were examined by Normarski optics no hairy cells were seen to form E-rosettes. These observations suggest that there are indeed two populations of lymphocytes (hairy and smooth) that can be identified by Normarski interference contrast microscopy, but they do not correspond with the population of thymus and bone-marrow derived lymphocytes as assayed by other markers. From our data it is reasonable to assume the E-rosette-forming cells are contained within the smooth lymphocyte population. The relationship between the surface topography of lymphocytes observed using this system and that seen using the critical-point drying technique and scanning electron microscopy remains to be clarified. For example, the almost uniform smooth-cell surface appearance in the 5 cases of C.L.L. that we observed contrasts with the observations of Polliak et al.,19 where cells showed numerous surface microvilli. This work was supported in part by U.S.P.H.S. grants CA-14502 and CA-1287 from the National Cancer Institute. Transplantation Immunology Laboratory and Division of Hematology, JOSE M. VILA Mount Sinai School of Medicine, ROBERT N. TAUB. New York, N.Y. 10029, U.S.A.
14.
18. Reyes, F., Lejonc, J. L., Gourdin, M. F., Mannoni, P., Dreyfus, B. J. exp. Med. 1975, 141, 392. 19. Polliak, A., Lampern, N., Clarkson, B. D., DeHarven, E., Bentwich L., Siegel, F. P., Kunkel, H. G. ibid. 1973, 138, 607.
RESULTS WITH NORMARSKI OPTICS AND WITH T AND B MARKERS
I
*
.
r
=
correlation coefficient.
,