S U R F A C E M A R K E R S O N L Y M P H O C Y T E S A N D C ELLS OF THE MONONUCLEAR PHAGOCYTE SERIES I N SKIN SECTIONS I N L E PRO SY MARIANJ. RIDLEYAND D. S. RIDLEY Hospital for Tropical Diseases, London N W l AND
J. L. TURK Royal College of Surgeons, London WC2
T H E histological classification of leprosy has been well worked out (Ridley 1974) and correlated with lymphocytic transformation test (LTT) results with circulating lymphocytes (Myrvang et al., 1973; Bjune et al., 1976; Job et al., 1976). Circulating lymphocytes have been further studied by rosette tests (Dwyer, Bullock and Fields, 1973; Nath et al., 1974; Lim et al., 1974; Sher et al., 1976) and the performance of macrophages from leprosy patients in vitro has been shown to be dependent on the presence of lymphocytes by Godal et al. (1971), Drutz, Cline and Levy (1974) and Parmaswaran et al. (1976). However, identification of cells by surface markers has not so far been applied to cells present in leprosy lesions. Ridley (1974) and Bjune et al. (1976) found that the presence of lymphocytes in lesions was the only criterion of histological classification that did not correlate well with the LTT. In the present investigation, rosette tests E, EA and EAC and other tests for surface receptors, have been applied to cryostat sections of skin biopsies in leprosy using techniques of Dukor, Bianco and Nussenweig (1970), Silveira, Mendes and Tolnai (1972) and Meijer and Lindeman (1975). Unexpectedly, the heaviest binding of EA and EAC was found to occur in parts of the granuloma where there were few lymphocytes and in the region of the epidermis and certain other skin structures. The results have not yet been fully interpreted. For comparison, tests with surface markers were carried out on a few skin lesions of other mycobacterial conditions, of sarcoidosis and of cutaneous leishmaniasis. MATERIAL AND
METHODS
Skin biopsies were obtained from 22 patients covering the TT-LL spectrum of leprosy (T = tuberculoid; L = lepromatous; B = borderline, Ridley and Jopling 1966), one TT, three BT and two BB (these were taken as a group for the purpose of analysis), four BL and eight LL (three active LLA, five in regression LLR). Four were in reaction, two with erythema nodosum leprosum (ENL) and two BT in reaction. In addition skin biopsies Received 4 Nov. 1977; accepted 10 Nov. 1977. J. PATH.-VOL.
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were studied from one patient with sarcoidosis, one atypical Mycobacterium ulcerans infection, one cutaneous leishmaniasis and two from skins which proved to be histologically normal. The lesion of sarcoidosis was from an untreated patient with a history of 6 yr duration affecting skin only. The Mycobacterium ulcerans infection was in the granulomatous phase, AFB were numerous. The leishmaniasis patient had been treated but not cured. Localisation of receptor sites was carried out on four serial sections in each case to establish reproducibility of results. Preparation of E cells. Fresh sheep cells in Alsever's Solution (E) were washed x 3 in Hanks' buffered salt solution (HBSS) at pH 7.4. A 3 per cent. suspension of cells was made for use. Preparation of EA. An aliquot of the 3 per cent. was taken and sensitised with rabbit anti-sheep (whole serum) antibody at 1/1500 (EA) at room temperature for 30 min. The EA was washed x 3 in HBSS, sedimented at 200 g for 10 min and resuspended to a final concentration of 3 per cent. cells. Preparation of EAC. An aliquot of EA was taken and coated with mouse complement diluted to a sub-lytic dose, usually 1/10. The suspension was incubated at 37" for 30 mins then washed x 3 in HBSS, sedimented at 200 g for 10 mins and resuspended to a final concentration of 3 per cent. Detection of tissue localisation. 6 pm cryostat sections were cut, air dried for a few minutes, washed in HBSS and transferred to a moist chamber. To demonstrate E, EA and EAC receptors, sections were incubated according to the method of Silveira et al. (1972). Three incubation schedules were tested: (1) at 37" for 30 min then 4" for 1 hr; (2) at room temperature for 1 hr; (3) at 37" for 30 min then 4" overnight. The slides were washed gently in HBSS until no E floated off. The solution for washing was kept at room temperature and cold buffer was used for those sections incubated at 4". The washed slides were fixed in 4 per cent. paraformaldehyde in cacodylate buffer at pH 7.4 for 3 hr. After fixing, the slides were washed and stained in haematoxylin-eosin. Blocking of EA and EAC. An aliquot of EAC was incubated with 1/20 dilution of antihuman C3 serum (Miles-Yeda) at 37" for 30 min. The EAC was washed, sedimented and resuspended for use. The EAC rosette test was then carried out on the sections as above. Trypsin was applied to the sections at a concentration of 0.1 mg/ml for 30 min at 37". Sections were washed in HBSS before the EAC test was carried out. Immunoglobulins. Immunofluorescent staining was done with purified anti-IgG, anti-IgM and anti-IgA (Burroughs-Wellcome) conjugated with FITC and diluted 1 /4. Incubation was 1 hr in a moist chamber at 4". Sections were washed in phosphate buffered saline (PBS) and examined in glycerol-phosphate buffer.
RESULTS
The schedule which was found to give the best results and which was adopted as standard was incubation of sections at room temperature for a period not exceeding 1 hr. E did not adhere to any of the constituents of skin sections and is hereafter disregarded. EA and EAC adhered either in uniform sheets or in clusters over or around groups of cells or skin structures. Rosettes were not seen around individual cells. Tests performed on serial sections gave entirely reproducible results. Lymphocytes
The results are summarised in Table I. In leprosy, lymphocytes were most numerous in the TT-BB group with a second peak in the BL group. When present in LL, lymphocytes were diffusely
CELL MARKERS IN LEPROSY
93
scattered over the macrophages. There was no EA or EAC adherence to lymphocytes of the TT-BB group. In BL there was EA and EAC adherence around lymphocytes. No adherence of EA or EAC was seen in the few lymphocytes of LL. TABLEI Numbers of lymphocytes and rosette adherence in skirr Iesioiis Leprosy
?T Lymphocytes E EA PAC
+ + ~
-
BB BL LLA R ' Sarcoid M. ulcerans Atyp. mycob. i -t 3-(diffuse) +-t ++$(focal) ++-k(focal) - - I + t -t I -1 f-ti I -I t+
BT
+-t t
+
+
CL tr. &(diffuse) -
t -i
No EA or EAC was seen in lymphocytes of sarcoidosis or in the tuberculin reaction, but in Mycobacterium ulcerans infection as in the atypical mycobacterial infection, clusters of lymphocytes in the deep tissues showed EA and EAC binding similar to that of BL leprosy. In the case of cutaneous leishmaniasis with a diffuse infiltrate of lymphocytes in the granuloma, there was minimal adherence of EA and EAC round lymphocytes.
Epithelioid cells and macrophages A most striking result was that the strongest adherence was seen not over lymphocytes but over cells of the mononuclear phagocytic system in skin. This applied particularly to EAC. There was a sharp distinction between macrophages and epithelioid cells in that macrophages showed both EA and EAC while epithelioid cells showed EAC only. In leprosy EAC was heaviest at the TT-BT end of the spectrum with a definite gradual diminution towards the LL end. EA adherence, on the other hand, showed a peak of intensity in the BL group with a decline towards BT and active LL. There was no EA adherence to foamy macrophages of LL in regression tables 11 and 111). TABLEI1 Adherence of cells of the mononirclear phagocy f ic system Leprosy
Reactions
In two cases of BT leprosy in reaction there was some EA and EAC adherence around macrophages, with a specially heavy EAC binding over epithelioid and giant cells. In ENL, EA and EAC binding was equally intense. The adherence was confined to the reactive areas with polymorph infiltration and was especially marked around the blood vessels. Bacilli were scanty at these sites although elsewhere in the section numerous bacilli were seen in I. PATH.-VOL.
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94
foamy cells. Notably in both cases of BT in reaction as well as in ENL, adherence was seen most strikingly in the intercellular spaces around cells of the mononuclear phagocyte system. In sarcoidosis there was a marked EAC adherence round epithelioid cells. This was in line with BT in reaction. In Mycobacterium ulcerans infection as in atypical mycobacterial infection, EA and EAC was marked throughout the granuloma. There was heavy adherence of EAC round blood vessels as in ENL. In cutaneous leishmaniasis there was some EA and EAC in macrophages of the granuloma which, however, was diminished in size as a result of previous trea trnent . TABLE111 Summary of spectrum of cells of mononuclear phagocytic system TT/RB
monocytes
I
I
EAC--
EA-
LL
young macrophages
monocy tes -1
EA-
I EACi
~.
EL1
I
I
EAC
1
young macrophages
I
r - - - i E A i iEAC t
OJ
epithelioid cells
I
EAC t -f -I
leprosy giant cells
I
EAC t t I-
foam cells
I
r--
EA-
E A C ~
Epidermis and skin stnirtures
The epidermis, sweat glands and hair follicles showed marked EAC adherence in the normal skin contents as well as those of the lesions studied. There was an increase of EAC in the oedematous hyperplastic epidermis of leprosy in reaction and a marked diminution in the skin of LL leprosy. Similarly there was loss of EAC adherence in some hair follicles of BT. Nerves
The binding of EA and EAC to epithelioid cells, macrophages and lymphocytes in and around nerve bundles in the skin followed much the same pattern as in other parts of the lesion. But whereas in the TT-BT region, in which the perineurium was fairly intact, the EAC was maximal in the centre of the nerve bundle, in BB where the macrophages and epithelioid cells were infiltrating through the perineurium the EAC was maximal in the perineurial zone. In BL, EA was maximal over the macrophages within the bundle. There was some binding of EA and EAC around the lymphocytes in the perneurium. In LL there were no macrophages or lymphocytes in the nerve or perineurium and there was no binding. In one case of BT in reaction in which there was Some fibrinoid necrosis in a nerve, the necrotic area produced intense binding. Immunoglobulins
The pattern of immunoglobulin fluorescence in cryostat sections in various types of disease is summarised in table IV. Tn general IgG correlated with EA
CELL MARKERS IN LEPROSY
95
binding and macrophages, IgM with EAC and epithelioid cells. Small amounts of IgA were detected in the Mycobacterium ulcerans lesion and in sarcoidosis, but not in leprosy. Blocking experimwts
Anti C3 diminished much of the EAC adherence in the granuloma but not in the epidermis. Trypsin partly blocked the EAC adherence in the granuloma and epidermis. EA adherence was removed by trypsin. TABLE1V Distributioii of inimunogiobiiliti iuavicers irt sectiuris of gra/iulomntous coiiditioiis Leprosy J.
f
Test IgG IgM
&A
BT-BB -
+ -
BL
+
-
LL i-
7
BTR $.
-
ENL -
+ -
Sarcoidosis Atyp. mycob. M. ulcerans CL 4-
t
-t
-
+++4
DISCUSSION
Like Edelson et al. (1973), we were unable to demonstrate T cells by E rosette in skin biopsies, although Claudy et al. (1976) and Meijer et al. (1977) were able to do so after dissociation from inflammatory tissue. E cell adherence is thought to be a physiological function unrelated to an immune mechanism and according to Coombs et al. (1970), produced only by living cells. Identification of T cells is by exclusion. In TT-BT leprosy the predominant infiltrating lymphocyte is here shown to be EA and EAC negative from which it may be inferred that these are T cells. The second peak of lymphocytic infiltration is in the BL group. Here the lymphocytes were seen in clusters. We identified these cells as B lymphocytes on account of EA and EAC adherence round individual cells and a positive JgG fluorescence in some cells. Their significance is yet to be determined but Ridley (1974) notes that their presence correlates with resistance to downgrading. Sher et al. (1976) suggest that the low B cell count in circulating lymphocytes may be due to trapping of these cells in the tissues. Lymphocytes in sarcoidosis and the tuberculin reaction were negative for EA and EAC which supports the conclusion of Tannebaum et al. (1976) that the cells in sarcoidosis are T cells. In mycobacterial infections lymphocytes were found to be B cell types as in BL leprosy. Two distinct patterns of EA and EAC adherence were seen in granulomas. The macrophages of active BL-LL leprosy were found to have both Fc and C3 receptors and at this stage were indistinguishable from those of BT-BB which had the same receptors. However in those macrophages of BL-LL which had phagocytosed M. lepvae, the C3 receptor site was lost and the Fc site reduced until, at the foamy cell stage, no receptor site could be demonstrated. Immunoglobulins were of the IgG class. This observation is in line with that of Huber
+
c -
96
MARIAN RIDLEY, D . RIDLEY A N D J. TURK
et al. (1968). On the other hand in TT-BT leprosy epithelioid cells showed a strong expression of the C3 receptor site. The significance of this finding needs further study. It is not clear from the EA and EAC adherence to granulomas whether we are dealing with Fc and C3 receptor sites alone or complexes of a similar configuration. Anti-C, and trypsin only partly blocked the adherence which could still be demonstrated, particularly in the intercellular spaces in some situations. It was most marked in BT in reaction and in Mycobact. ulcerans lesions, in both of which there was intercellular oedema. IgM was present at the same sites, and the combinations of this with EAC was thought to signify immune complexes. In ENL the distribution of immune complexes on the periphery of macrophage areas with a low bacterial load, demonstrated by Wemambu et al. (1969), supports the concept of an Arthus type reaction which occurs only in situations of antibody excess. But immune complexes have not previously been demonstrated in leprosy reactions of the borderline type. Waaler et a/. (1971) using a " mixed agglutination reaction " with a technique essentially the same as that of our own EA test, obtained positives in 31 of 51 skin biopsies of leprosy. The results (which we had not seen at the time we carried out our own study) are almost identical to our EA results, since their tests were positive only in lepromatous cases during the active phase of the infection or in ENL. Waaler et al. interpret this as indicative of anti-y-globulin activity in the lesions, with the characteristics of rheumatoid factor, though the results did not correlate with rheumatoid factor in the serum. This seems to us much less likely than our explanation of binding to FC receptor sites. EAC binding is consistently maximal in reactions in borderline leprosy, which are presumed to be of the delayed hypersensitivity type. In nerves, EAC was maximal in two situations : (i) in the centre of the nerve bundle in BT, which is the position in which necrosis is most likely to occur, and (ii) in the perineurial zone in BB, in which epithelioid cells are penetrating the perineurium, which becomes laminated. The evidence is inconclusive, but these findings would be consistent with the possibility that tissue damage occurs in nerves when M. leprae is first recognised by the immune mechanism, and that the sites where this is most likely to occur are the central zone when immunity is moderately high, and the perineurium when the immunity is lower and bacilli are not recognised until they have multiplied to the point at which they are able to break out from the nerve bundle. Boddinguis (1977) finds evidence that the damage to the perineurium comes about through the presence of M. leprae in the epi-, peri and endoneurial blood vessels. Positive EAC adherance to epidermis which cannot be blocked by anti-C3 has yet to be elucidated. Epithelial cells could be concerned with the processing of complement. The diminution in LL is probably due to the atrophy of the epidermis because the epithelial cells of skin structures in the same lesion give a normal response. Similarly the atrophic hair follicles of TT did not produce EAC adherence.
CELL MARKERS IN LEPROSY
97
SUMMARY
E, EA and EAC rosetting techniques and Ig fluorescence were used in a study of receptor sites in cryostat sections of lesions through the spectrum of leprosy, and for comparison in some other mycobacterial and granulomatous lesions. AntiC3, and trypsin were used as blocking agents. Lymphocytes in borderline lepromatous leprosy produced EA adherence and IgG fluorescence indicating B type cells. Lymphocytes in tuberculoid leprosy produced neither E or EA adherence and no fluorescence; these cells were presumed to be T cells. EAC and EA adherence was more marked in areas of macrophage infiltration, where there were few lymphocytes, than over the lympocytes themselves. Two distinct patterns emerged: (i) EA binding together with IgG fluorescence was seen in active lepromatous leprosy and could be localised to the surface of individual macrophages, and (ii) EAC binding together with JgM fluorescence was seen in the granuloma of tuberculoid leprosy and sarcoidosis, but could not be definitely related to cell surface; rather it was diffusely spread over the whole granuloma; EAC adherence was diminished by anti-C3 serum. Trypsin removed EA binding completely, but only diminished EAC adherence. It is suggested that the EA pattern indicates immunoglobulin receptors on macrophage and lymphocyte surfaces: and that the EAC binding (which is stronger than EA) involves C3 and IgM receptors at extracellular sites as well as C3 receptor sites on epithelioid cell surfaces. EA and EAC binding were enhanced in borderline tuberculoid leprosy in reaction and erythema nodosum leprosum, suggesting that immunoglobulin and complement receptor sites increase in number with enhanced hypersensitivity. REFERENCES BODDINGIUS, J. 1977. Ultra structural changes in blood vessels of peripheral nerves in leprosy Neuropathy. TI. Borderline, borderline-lepromatous and lepromatous leprosy patients. Acrn Neuropath. (Berl), 40, 21-39. BJUNE,G., BAKNETSON, R. ST C., RIDLEY,D. s.,AND KKONVALL, G. 1976. Lymphocyte transformation test in leprosy; Correlation of response with inflammation of lesions. Clin. exp. Immunol., 25, 85-94. CLAUDY, A. L., SCHMITT, D., VIAC,J., ALAIRO, A., STAQUET, M. J., AND THIVOLET, J. 1976. Morphological, immunological and iinmunocytochemical identification of lymphocytes extracted from cutaneous infiltrates. Clin. exp. Immunol., 23, 61-68. COOMBS, R. R. A., GUKNER, B. N., WILSON,A. B., HOLM,G., AND LINDGREN, B. 1970. Rosette formation between human lymphocytes and sheep red cells not involving immunoglobulin receptors. int. Arch. Allergy and Appl. Immunol., 39, 658-663. DRUTZ,D. J., CLINE, M. J., AND LEVY,L. 1974. Leukocyte antimicrobial function in patients with leprosy. J. clin. inveht., 53, 380-386. DUKOR,P., BIANCO,C., AND NUSSENWEIG, V. 1970. Tissue localisation of lymphocytes bearing a receptor for antigen antibody complement complexes. Proc. Nut. Acad. Sci. USA, 67,991-997. DWYER,J. M., BULLOCK,W. E., AND FIELDS,J. P. 1973. Disturbance of blood T : B lymphocyte ratio in lepromatous leprosy. New Eng. J. Med., 228, 1036-1039.
9s
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EDELSON, R. L., SMITH,E. W., FRANK, M. U. N., AND GREEN, J. 1973. Identification of subpopulations of mononuclear cells in cutaneous infiltrates. Difference between T and B cells and histocytes. J. invest. Derm., 61, 82. GODAL,T., REES,R. J. W., AND LAMVIK, JO. 1971. Lymphocyte mediated modification of blood derived macrophage function in vitro including inhibition of growth of intra cellular mycobacteria. Clin. exp. Immunol., 8, 625-637. HUBER,H., POLLEY, M. J., LINSCOTT, W. D., AND MULLER-EBERHARD, H. J. 1968. Human monocytes: Distinct receptor sites for the third component of complement and for immunoglobulin G. Science, 162, 1281-1283. JOB,C. K., CHACKO, C. J. G., TAYLOR, P. M., DANIEL, M., AND JESUDIAN, G. 1976. Evaluation of Cell Mediated Immunity in the histopathologic spectrum of leprosy using lymphocyte transformation test. International Journal of Leprosy, 44,256-266. LTM,S. D., KISZKISS, D. F., JACOBSON, R. R., CHOI,Y. S., GOOD,R. A. 1974. Thymus dependent lymphocytes of peripheral blood in leprosy patients. InJ Immunity, 9, 394. MEIJER,C. J. L. M., AND LINDEMAN, J. 1975. A modified method for tissue localisation of cells bearing a complement receptor. J. Immunol. Meth., 9, 59-65. MEIJER,C. J. L. M., VANDE FUTTE,L. B. A., EULDERINK, F., KLEINJAN, R., LAFEBER, G., AND BOTS,G. TH. A. M. 1977. Characteristics of mononuclear cell populations in chronically inflamed synovial membranes. J. Path., 121, 1-8. MYRVANG, B., GODAL,T.,RIDLEY,D. s.,FR0LAND, s. s.,AND SONG, Y. K. 1973. Immune responsiveness to mycobacterium leprae and other mycobacterial antigens throughout the clinical and histological spectrum of leprosy. Clin. exp. Immuiiol., 14, 541-553. NATH,I., CURTIS,J., BHUTANI, L. K., AND TALWAR, G. P. 1974. Reduction of a subpopulation of T lymphocytes in lepromatous leprosy. Clin. exp. Immunol., 16, 23-30. PARMASWARAN, M., GIRDHAR, B. K., DEO,M. G., KANDHARI, K. C., AND BHUTANI, L. K. 1976. Macrophage Function in Leprosy. International Journal of Leprosy, 340-345. RIDLEY,D. S., AND JOPLING, W. H. 1962. A classification of leprosy for research purposes. Lepr. Rev. 33, 119-128. RIDLEY,D. S. 1974. Histological classification and the immunological spectrum of leprosy. Bull. WId. Hlth. Org. SHER,R., HOLM,G., KOK,S. H., KOORNHOF, H. F., AND GLOVER, F., T and CR+ Iymphocytic profile in leprosy and the effect fo treatment. In6 Immunity, 13, 31-35. SILVEIRA, N. P. A., MENDES, N. F., AND TOLNAI, M. E. A. 1972. Tissue localisation of 2 populations of human lymphocytes distinguished by membrane receptors. J. Zmmurzol., 108, 1456-1460. TANNEBAUM, H., ROCKLIN, R. E., SCHUR, P. H., AND SHEFFER, A. L. 1976. Immunefunction in sarcoidosis. Clin.exp. Immunol., 26, 51 1-519. WAALER, E., TONDER, D., GODAL, T., MILDE,E.-J. SOGHER, F., AND SHESKIN, J., 1971. Antiy-globulin activity in leprous lesions. Internnt. J . Leprosy, 39, 529-540. WEMAMBU, S. N. C., TURK,J. L., WATERS, M. F. R., AND REES,R. J. W. 1969. Erythema nodosum leprosum. A clinical manifestation of Arthus phenomenon. Lancet, ii, 933-935.
J. L. TURK Royal College of Surgeons, London WC2
T H E histological classification of leprosy has been well worked out (Ridley 1974) and correlated with lymphocytic transformation test (LTT) results with circulating lymphocytes (Myrvang et al., 1973; Bjune et al., 1976; Job et al., 1976). Circulating lymphocytes have been further studied by rosette tests (Dwyer, Bullock and Fields, 1973; Nath et al., 1974; Lim et al., 1974; Sher et al., 1976) and the performance of macrophages from leprosy patients in vitro has been shown to be dependent on the presence of lymphocytes by Godal et al. (1971), Drutz, Cline and Levy (1974) and Parmaswaran et al. (1976). However, identification of cells by surface markers has not so far been applied to cells present in leprosy lesions. Ridley (1974) and Bjune et al. (1976) found that the presence of lymphocytes in lesions was the only criterion of histological classification that did not correlate well with the LTT. In the present investigation, rosette tests E, EA and EAC and other tests for surface receptors, have been applied to cryostat sections of skin biopsies in leprosy using techniques of Dukor, Bianco and Nussenweig (1970), Silveira, Mendes and Tolnai (1972) and Meijer and Lindeman (1975). Unexpectedly, the heaviest binding of EA and EAC was found to occur in parts of the granuloma where there were few lymphocytes and in the region of the epidermis and certain other skin structures. The results have not yet been fully interpreted. For comparison, tests with surface markers were carried out on a few skin lesions of other mycobacterial conditions, of sarcoidosis and of cutaneous leishmaniasis. MATERIAL AND
METHODS
Skin biopsies were obtained from 22 patients covering the TT-LL spectrum of leprosy (T = tuberculoid; L = lepromatous; B = borderline, Ridley and Jopling 1966), one TT, three BT and two BB (these were taken as a group for the purpose of analysis), four BL and eight LL (three active LLA, five in regression LLR). Four were in reaction, two with erythema nodosum leprosum (ENL) and two BT in reaction. In addition skin biopsies Received 4 Nov. 1977; accepted 10 Nov. 1977. J. PATH.-VOL.
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H
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MARIAN RIDLEY, D. RIDLEY AND J. TURK
were studied from one patient with sarcoidosis, one atypical Mycobacterium ulcerans infection, one cutaneous leishmaniasis and two from skins which proved to be histologically normal. The lesion of sarcoidosis was from an untreated patient with a history of 6 yr duration affecting skin only. The Mycobacterium ulcerans infection was in the granulomatous phase, AFB were numerous. The leishmaniasis patient had been treated but not cured. Localisation of receptor sites was carried out on four serial sections in each case to establish reproducibility of results. Preparation of E cells. Fresh sheep cells in Alsever's Solution (E) were washed x 3 in Hanks' buffered salt solution (HBSS) at pH 7.4. A 3 per cent. suspension of cells was made for use. Preparation of EA. An aliquot of the 3 per cent. was taken and sensitised with rabbit anti-sheep (whole serum) antibody at 1/1500 (EA) at room temperature for 30 min. The EA was washed x 3 in HBSS, sedimented at 200 g for 10 min and resuspended to a final concentration of 3 per cent. cells. Preparation of EAC. An aliquot of EA was taken and coated with mouse complement diluted to a sub-lytic dose, usually 1/10. The suspension was incubated at 37" for 30 mins then washed x 3 in HBSS, sedimented at 200 g for 10 mins and resuspended to a final concentration of 3 per cent. Detection of tissue localisation. 6 pm cryostat sections were cut, air dried for a few minutes, washed in HBSS and transferred to a moist chamber. To demonstrate E, EA and EAC receptors, sections were incubated according to the method of Silveira et al. (1972). Three incubation schedules were tested: (1) at 37" for 30 min then 4" for 1 hr; (2) at room temperature for 1 hr; (3) at 37" for 30 min then 4" overnight. The slides were washed gently in HBSS until no E floated off. The solution for washing was kept at room temperature and cold buffer was used for those sections incubated at 4". The washed slides were fixed in 4 per cent. paraformaldehyde in cacodylate buffer at pH 7.4 for 3 hr. After fixing, the slides were washed and stained in haematoxylin-eosin. Blocking of EA and EAC. An aliquot of EAC was incubated with 1/20 dilution of antihuman C3 serum (Miles-Yeda) at 37" for 30 min. The EAC was washed, sedimented and resuspended for use. The EAC rosette test was then carried out on the sections as above. Trypsin was applied to the sections at a concentration of 0.1 mg/ml for 30 min at 37". Sections were washed in HBSS before the EAC test was carried out. Immunoglobulins. Immunofluorescent staining was done with purified anti-IgG, anti-IgM and anti-IgA (Burroughs-Wellcome) conjugated with FITC and diluted 1 /4. Incubation was 1 hr in a moist chamber at 4". Sections were washed in phosphate buffered saline (PBS) and examined in glycerol-phosphate buffer.
RESULTS
The schedule which was found to give the best results and which was adopted as standard was incubation of sections at room temperature for a period not exceeding 1 hr. E did not adhere to any of the constituents of skin sections and is hereafter disregarded. EA and EAC adhered either in uniform sheets or in clusters over or around groups of cells or skin structures. Rosettes were not seen around individual cells. Tests performed on serial sections gave entirely reproducible results. Lymphocytes
The results are summarised in Table I. In leprosy, lymphocytes were most numerous in the TT-BB group with a second peak in the BL group. When present in LL, lymphocytes were diffusely
CELL MARKERS IN LEPROSY
93
scattered over the macrophages. There was no EA or EAC adherence to lymphocytes of the TT-BB group. In BL there was EA and EAC adherence around lymphocytes. No adherence of EA or EAC was seen in the few lymphocytes of LL. TABLEI Numbers of lymphocytes and rosette adherence in skirr Iesioiis Leprosy
?T Lymphocytes E EA PAC
+ + ~
-
BB BL LLA R ' Sarcoid M. ulcerans Atyp. mycob. i -t 3-(diffuse) +-t ++$(focal) ++-k(focal) - - I + t -t I -1 f-ti I -I t+
BT
+-t t
+
+
CL tr. &(diffuse) -
t -i
No EA or EAC was seen in lymphocytes of sarcoidosis or in the tuberculin reaction, but in Mycobacterium ulcerans infection as in the atypical mycobacterial infection, clusters of lymphocytes in the deep tissues showed EA and EAC binding similar to that of BL leprosy. In the case of cutaneous leishmaniasis with a diffuse infiltrate of lymphocytes in the granuloma, there was minimal adherence of EA and EAC round lymphocytes.
Epithelioid cells and macrophages A most striking result was that the strongest adherence was seen not over lymphocytes but over cells of the mononuclear phagocytic system in skin. This applied particularly to EAC. There was a sharp distinction between macrophages and epithelioid cells in that macrophages showed both EA and EAC while epithelioid cells showed EAC only. In leprosy EAC was heaviest at the TT-BT end of the spectrum with a definite gradual diminution towards the LL end. EA adherence, on the other hand, showed a peak of intensity in the BL group with a decline towards BT and active LL. There was no EA adherence to foamy macrophages of LL in regression tables 11 and 111). TABLEI1 Adherence of cells of the mononirclear phagocy f ic system Leprosy
Reactions
In two cases of BT leprosy in reaction there was some EA and EAC adherence around macrophages, with a specially heavy EAC binding over epithelioid and giant cells. In ENL, EA and EAC binding was equally intense. The adherence was confined to the reactive areas with polymorph infiltration and was especially marked around the blood vessels. Bacilli were scanty at these sites although elsewhere in the section numerous bacilli were seen in I. PATH.-VOL.
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H*
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94
foamy cells. Notably in both cases of BT in reaction as well as in ENL, adherence was seen most strikingly in the intercellular spaces around cells of the mononuclear phagocyte system. In sarcoidosis there was a marked EAC adherence round epithelioid cells. This was in line with BT in reaction. In Mycobacterium ulcerans infection as in atypical mycobacterial infection, EA and EAC was marked throughout the granuloma. There was heavy adherence of EAC round blood vessels as in ENL. In cutaneous leishmaniasis there was some EA and EAC in macrophages of the granuloma which, however, was diminished in size as a result of previous trea trnent . TABLE111 Summary of spectrum of cells of mononuclear phagocytic system TT/RB
monocytes
I
I
EAC--
EA-
LL
young macrophages
monocy tes -1
EA-
I EACi
~.
EL1
I
I
EAC
1
young macrophages
I
r - - - i E A i iEAC t
OJ
epithelioid cells
I
EAC t -f -I
leprosy giant cells
I
EAC t t I-
foam cells
I
r--
EA-
E A C ~
Epidermis and skin stnirtures
The epidermis, sweat glands and hair follicles showed marked EAC adherence in the normal skin contents as well as those of the lesions studied. There was an increase of EAC in the oedematous hyperplastic epidermis of leprosy in reaction and a marked diminution in the skin of LL leprosy. Similarly there was loss of EAC adherence in some hair follicles of BT. Nerves
The binding of EA and EAC to epithelioid cells, macrophages and lymphocytes in and around nerve bundles in the skin followed much the same pattern as in other parts of the lesion. But whereas in the TT-BT region, in which the perineurium was fairly intact, the EAC was maximal in the centre of the nerve bundle, in BB where the macrophages and epithelioid cells were infiltrating through the perineurium the EAC was maximal in the perineurial zone. In BL, EA was maximal over the macrophages within the bundle. There was some binding of EA and EAC around the lymphocytes in the perneurium. In LL there were no macrophages or lymphocytes in the nerve or perineurium and there was no binding. In one case of BT in reaction in which there was Some fibrinoid necrosis in a nerve, the necrotic area produced intense binding. Immunoglobulins
The pattern of immunoglobulin fluorescence in cryostat sections in various types of disease is summarised in table IV. Tn general IgG correlated with EA
CELL MARKERS IN LEPROSY
95
binding and macrophages, IgM with EAC and epithelioid cells. Small amounts of IgA were detected in the Mycobacterium ulcerans lesion and in sarcoidosis, but not in leprosy. Blocking experimwts
Anti C3 diminished much of the EAC adherence in the granuloma but not in the epidermis. Trypsin partly blocked the EAC adherence in the granuloma and epidermis. EA adherence was removed by trypsin. TABLE1V Distributioii of inimunogiobiiliti iuavicers irt sectiuris of gra/iulomntous coiiditioiis Leprosy J.
f
Test IgG IgM
&A
BT-BB -
+ -
BL
+
-
LL i-
7
BTR $.
-
ENL -
+ -
Sarcoidosis Atyp. mycob. M. ulcerans CL 4-
t
-t
-
+++4
DISCUSSION
Like Edelson et al. (1973), we were unable to demonstrate T cells by E rosette in skin biopsies, although Claudy et al. (1976) and Meijer et al. (1977) were able to do so after dissociation from inflammatory tissue. E cell adherence is thought to be a physiological function unrelated to an immune mechanism and according to Coombs et al. (1970), produced only by living cells. Identification of T cells is by exclusion. In TT-BT leprosy the predominant infiltrating lymphocyte is here shown to be EA and EAC negative from which it may be inferred that these are T cells. The second peak of lymphocytic infiltration is in the BL group. Here the lymphocytes were seen in clusters. We identified these cells as B lymphocytes on account of EA and EAC adherence round individual cells and a positive JgG fluorescence in some cells. Their significance is yet to be determined but Ridley (1974) notes that their presence correlates with resistance to downgrading. Sher et al. (1976) suggest that the low B cell count in circulating lymphocytes may be due to trapping of these cells in the tissues. Lymphocytes in sarcoidosis and the tuberculin reaction were negative for EA and EAC which supports the conclusion of Tannebaum et al. (1976) that the cells in sarcoidosis are T cells. In mycobacterial infections lymphocytes were found to be B cell types as in BL leprosy. Two distinct patterns of EA and EAC adherence were seen in granulomas. The macrophages of active BL-LL leprosy were found to have both Fc and C3 receptors and at this stage were indistinguishable from those of BT-BB which had the same receptors. However in those macrophages of BL-LL which had phagocytosed M. lepvae, the C3 receptor site was lost and the Fc site reduced until, at the foamy cell stage, no receptor site could be demonstrated. Immunoglobulins were of the IgG class. This observation is in line with that of Huber
+
c -
96
MARIAN RIDLEY, D . RIDLEY A N D J. TURK
et al. (1968). On the other hand in TT-BT leprosy epithelioid cells showed a strong expression of the C3 receptor site. The significance of this finding needs further study. It is not clear from the EA and EAC adherence to granulomas whether we are dealing with Fc and C3 receptor sites alone or complexes of a similar configuration. Anti-C, and trypsin only partly blocked the adherence which could still be demonstrated, particularly in the intercellular spaces in some situations. It was most marked in BT in reaction and in Mycobact. ulcerans lesions, in both of which there was intercellular oedema. IgM was present at the same sites, and the combinations of this with EAC was thought to signify immune complexes. In ENL the distribution of immune complexes on the periphery of macrophage areas with a low bacterial load, demonstrated by Wemambu et al. (1969), supports the concept of an Arthus type reaction which occurs only in situations of antibody excess. But immune complexes have not previously been demonstrated in leprosy reactions of the borderline type. Waaler et a/. (1971) using a " mixed agglutination reaction " with a technique essentially the same as that of our own EA test, obtained positives in 31 of 51 skin biopsies of leprosy. The results (which we had not seen at the time we carried out our own study) are almost identical to our EA results, since their tests were positive only in lepromatous cases during the active phase of the infection or in ENL. Waaler et al. interpret this as indicative of anti-y-globulin activity in the lesions, with the characteristics of rheumatoid factor, though the results did not correlate with rheumatoid factor in the serum. This seems to us much less likely than our explanation of binding to FC receptor sites. EAC binding is consistently maximal in reactions in borderline leprosy, which are presumed to be of the delayed hypersensitivity type. In nerves, EAC was maximal in two situations : (i) in the centre of the nerve bundle in BT, which is the position in which necrosis is most likely to occur, and (ii) in the perineurial zone in BB, in which epithelioid cells are penetrating the perineurium, which becomes laminated. The evidence is inconclusive, but these findings would be consistent with the possibility that tissue damage occurs in nerves when M. leprae is first recognised by the immune mechanism, and that the sites where this is most likely to occur are the central zone when immunity is moderately high, and the perineurium when the immunity is lower and bacilli are not recognised until they have multiplied to the point at which they are able to break out from the nerve bundle. Boddinguis (1977) finds evidence that the damage to the perineurium comes about through the presence of M. leprae in the epi-, peri and endoneurial blood vessels. Positive EAC adherance to epidermis which cannot be blocked by anti-C3 has yet to be elucidated. Epithelial cells could be concerned with the processing of complement. The diminution in LL is probably due to the atrophy of the epidermis because the epithelial cells of skin structures in the same lesion give a normal response. Similarly the atrophic hair follicles of TT did not produce EAC adherence.
CELL MARKERS IN LEPROSY
97
SUMMARY
E, EA and EAC rosetting techniques and Ig fluorescence were used in a study of receptor sites in cryostat sections of lesions through the spectrum of leprosy, and for comparison in some other mycobacterial and granulomatous lesions. AntiC3, and trypsin were used as blocking agents. Lymphocytes in borderline lepromatous leprosy produced EA adherence and IgG fluorescence indicating B type cells. Lymphocytes in tuberculoid leprosy produced neither E or EA adherence and no fluorescence; these cells were presumed to be T cells. EAC and EA adherence was more marked in areas of macrophage infiltration, where there were few lymphocytes, than over the lympocytes themselves. Two distinct patterns emerged: (i) EA binding together with IgG fluorescence was seen in active lepromatous leprosy and could be localised to the surface of individual macrophages, and (ii) EAC binding together with JgM fluorescence was seen in the granuloma of tuberculoid leprosy and sarcoidosis, but could not be definitely related to cell surface; rather it was diffusely spread over the whole granuloma; EAC adherence was diminished by anti-C3 serum. Trypsin removed EA binding completely, but only diminished EAC adherence. It is suggested that the EA pattern indicates immunoglobulin receptors on macrophage and lymphocyte surfaces: and that the EAC binding (which is stronger than EA) involves C3 and IgM receptors at extracellular sites as well as C3 receptor sites on epithelioid cell surfaces. EA and EAC binding were enhanced in borderline tuberculoid leprosy in reaction and erythema nodosum leprosum, suggesting that immunoglobulin and complement receptor sites increase in number with enhanced hypersensitivity. REFERENCES BODDINGIUS, J. 1977. Ultra structural changes in blood vessels of peripheral nerves in leprosy Neuropathy. TI. Borderline, borderline-lepromatous and lepromatous leprosy patients. Acrn Neuropath. (Berl), 40, 21-39. BJUNE,G., BAKNETSON, R. ST C., RIDLEY,D. s.,AND KKONVALL, G. 1976. Lymphocyte transformation test in leprosy; Correlation of response with inflammation of lesions. Clin. exp. Immunol., 25, 85-94. CLAUDY, A. L., SCHMITT, D., VIAC,J., ALAIRO, A., STAQUET, M. J., AND THIVOLET, J. 1976. Morphological, immunological and iinmunocytochemical identification of lymphocytes extracted from cutaneous infiltrates. Clin. exp. Immunol., 23, 61-68. COOMBS, R. R. A., GUKNER, B. N., WILSON,A. B., HOLM,G., AND LINDGREN, B. 1970. Rosette formation between human lymphocytes and sheep red cells not involving immunoglobulin receptors. int. Arch. Allergy and Appl. Immunol., 39, 658-663. DRUTZ,D. J., CLINE, M. J., AND LEVY,L. 1974. Leukocyte antimicrobial function in patients with leprosy. J. clin. inveht., 53, 380-386. DUKOR,P., BIANCO,C., AND NUSSENWEIG, V. 1970. Tissue localisation of lymphocytes bearing a receptor for antigen antibody complement complexes. Proc. Nut. Acad. Sci. USA, 67,991-997. DWYER,J. M., BULLOCK,W. E., AND FIELDS,J. P. 1973. Disturbance of blood T : B lymphocyte ratio in lepromatous leprosy. New Eng. J. Med., 228, 1036-1039.
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EDELSON, R. L., SMITH,E. W., FRANK, M. U. N., AND GREEN, J. 1973. Identification of subpopulations of mononuclear cells in cutaneous infiltrates. Difference between T and B cells and histocytes. J. invest. Derm., 61, 82. GODAL,T., REES,R. J. W., AND LAMVIK, JO. 1971. Lymphocyte mediated modification of blood derived macrophage function in vitro including inhibition of growth of intra cellular mycobacteria. Clin. exp. Immunol., 8, 625-637. HUBER,H., POLLEY, M. J., LINSCOTT, W. D., AND MULLER-EBERHARD, H. J. 1968. Human monocytes: Distinct receptor sites for the third component of complement and for immunoglobulin G. Science, 162, 1281-1283. JOB,C. K., CHACKO, C. J. G., TAYLOR, P. M., DANIEL, M., AND JESUDIAN, G. 1976. Evaluation of Cell Mediated Immunity in the histopathologic spectrum of leprosy using lymphocyte transformation test. International Journal of Leprosy, 44,256-266. LTM,S. D., KISZKISS, D. F., JACOBSON, R. R., CHOI,Y. S., GOOD,R. A. 1974. Thymus dependent lymphocytes of peripheral blood in leprosy patients. InJ Immunity, 9, 394. MEIJER,C. J. L. M., AND LINDEMAN, J. 1975. A modified method for tissue localisation of cells bearing a complement receptor. J. Immunol. Meth., 9, 59-65. MEIJER,C. J. L. M., VANDE FUTTE,L. B. A., EULDERINK, F., KLEINJAN, R., LAFEBER, G., AND BOTS,G. TH. A. M. 1977. Characteristics of mononuclear cell populations in chronically inflamed synovial membranes. J. Path., 121, 1-8. MYRVANG, B., GODAL,T.,RIDLEY,D. s.,FR0LAND, s. s.,AND SONG, Y. K. 1973. Immune responsiveness to mycobacterium leprae and other mycobacterial antigens throughout the clinical and histological spectrum of leprosy. Clin. exp. Immuiiol., 14, 541-553. NATH,I., CURTIS,J., BHUTANI, L. K., AND TALWAR, G. P. 1974. Reduction of a subpopulation of T lymphocytes in lepromatous leprosy. Clin. exp. Immunol., 16, 23-30. PARMASWARAN, M., GIRDHAR, B. K., DEO,M. G., KANDHARI, K. C., AND BHUTANI, L. K. 1976. Macrophage Function in Leprosy. International Journal of Leprosy, 340-345. RIDLEY,D. S., AND JOPLING, W. H. 1962. A classification of leprosy for research purposes. Lepr. Rev. 33, 119-128. RIDLEY,D. S. 1974. Histological classification and the immunological spectrum of leprosy. Bull. WId. Hlth. Org. SHER,R., HOLM,G., KOK,S. H., KOORNHOF, H. F., AND GLOVER, F., T and CR+ Iymphocytic profile in leprosy and the effect fo treatment. In6 Immunity, 13, 31-35. SILVEIRA, N. P. A., MENDES, N. F., AND TOLNAI, M. E. A. 1972. Tissue localisation of 2 populations of human lymphocytes distinguished by membrane receptors. J. Zmmurzol., 108, 1456-1460. TANNEBAUM, H., ROCKLIN, R. E., SCHUR, P. H., AND SHEFFER, A. L. 1976. Immunefunction in sarcoidosis. Clin.exp. Immunol., 26, 51 1-519. WAALER, E., TONDER, D., GODAL, T., MILDE,E.-J. SOGHER, F., AND SHESKIN, J., 1971. Antiy-globulin activity in leprous lesions. Internnt. J . Leprosy, 39, 529-540. WEMAMBU, S. N. C., TURK,J. L., WATERS, M. F. R., AND REES,R. J. W. 1969. Erythema nodosum leprosum. A clinical manifestation of Arthus phenomenon. Lancet, ii, 933-935.
