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The pathogenesis of cutaneous leishmaniasis
150 TRANSACTIONS OP THB ROYAL Socmn
ROYAL
OP TROPICAL MEDICINE AND HYGIENE, VOL. 73, No. 2, 1979
SOCIETY
OF TROI’ICAI.
Ordinary
MEDICINE
Meeting
Manson House, Thursday,
AND HYGIENE
’
18th May, 1978
The President: DR. S. G. BROWNE in the Chair
The pathogenesis
of cutaneous
leishmaniasis
D. S., RIDLEY
Hospital for Tropical Diseases,London NW1 OPE The object of the present study is to have a look at the histological spectrum of cutaneous leishmaniasis (CL) in the hope that it might throw some light on the immunological mechanisms at work. The ultimate object would be to produce a classification that indicated prognosis, though this has not yet been achieved. The snectrum of CL, which ranges from the anergic to-the ultra-hypersensitive (;ecidiva) forms, has been compared-to the spectrum of lenrosv (DESTOMBES. 1960; TURK & BRYCESON, 19711. The-difference is that h CL it is the middle of the spectrum that produces the self-healing forms of disease, the two extremes both being non-selfhealing (BRYCESON, 1972). The inability of many patients to cure themselves without long delay, after having successfully eliminated parasites to the point where they are barely detectable, is perplexing. In the present paper, therefore, an attempt is made to relate pathology to immune mechanisms, and to compare -tie results with those in leprosy. The two diseases are found to be fundamentally different despite their many points in common. -
Patients
and methods
The varieties of CL are due not only to differences in immunological response but to species of parasite. To obtain an over-all view of the subject it was necessary to have biopsies from widespread sources. The present results relate mainly to a group of 33 cases of the purely cutaneous form of leishmaniasis seen at the Hospital for Tropical Diseases which represent all the main endemic areas of the world except for two special groups which were considered separately: (i) biopsies from 27 cases from Ethiopia, most of them sent bv Dr. R. StC. Barnetson, and (ii) biopsies from 22 mucocutaneous cases -from Brazil sent to me bv Dr. A. C. Barretto. Dr. C. C. Cuba and Dr. P. D. Marsden. I am most grateful to all these colleagues, including those in London, not only for the biopsies but for essential clinical data. All cases were active at the time of biopsy. In addition to routine histology, various histological characteristics were assessed quantitatively in a manner similar to that used previously in leprosy (BJUNE et al., 1976). These characteristics were numbers of lymphocytes and plasma cells, the development of granulomata both organised and unorganized, and necrosis. The scores were correlated with the parasite load, which was assessed on an approximately logarithmic parasite index (PI).
For an average sized lesion (approximately 2 x 10 objective fields), the scale was as follows: 5 + = ;;stytimerous amastigotes throughout the 4+
= numerous amastigotes in most parts of the lesion 3+ = numerous in a small part of the lesion (> 300 organisms) 2f = single focus of over 30 organisms l+ = single focus of under 30 organisms & = detected in smear, not biopsy -= not detectable in smear. An important practical point was the unreliability of biopsies of ulcers that did not include either the floor or the margin of the ulcer, though reluctance to take such biopsies was understandable. Most biopsies were good, a few were rejected. A well stained, fairly dark, haematoxylin-eosin was found to be the most satisfactory routine stain, better than Giemsa. Other stains were useful in selected cases.
The essential difference from leprosy The histological spectrum of leprosy (RIDLEY, 1974) depends on a number of features, but there is one that can be relied upon in every case. The character of the granuloma from macrophage to epithelioid and giant cell is invariably characteristic of the position in the spectrum. In CL it was found not to be so. When organisms were numerous the cell type nearly always was a macrophage, but when organisms were scanty the character of the granuloma was unpredictable; often there was none present. The granuloma is not the primary histological feature of CL.
Granulomata
(organized and unorganized)
It is necessary to digress to consider another important aspect of the granulomata of CL. There is only one pathway from macrophage to epithelioid and giant cell and it is the same whether the stimulus is the irritating qualities of the agent or the hypersensitivitv of the patient. The character of the granuloma composed of these same cell types is more varied. EPSTEIN (1967.1977) refers to granulomata as organized or unorganized, and he regards organized granulomata as signifying hypersensitivity. Whether this is always true is not certain, but one can see that hypersensitivity, by producing a strong stimulus operative at some distance from an agent, will help to organize cells in a radius around it. The
151
D. S. RIDLEY
Fig. 1. Organized granuloma suggesting hypersensitivity. L. donovani infection rapidly. (By courtesy of Prof. D. J. Bradley). H & E x 1500.
Fig. 2. Early granuloma
formation
(centre) on edge of necrotic
of liver in strain of mouse that eliminated
area (top left). Cutaneous
leishmtiasis.
parasites relatively
H & E X 215.
152
Fig. 3. More fully H & E Y 235.
PATHOGENESIS
developed
granuloma,
still
unorganized.
OF CUTANEOUS
It probably
small granulomata produced by Leishmania donovani in mouse liver (BRADLEY & KIRKLEY, 1977) is a good example of such organization (Fig. 1). More contentious is Epstein’s view that “granulomatous hypersensitivity” as represented here, is a specific entity of unknown nature. In many mycobacterial diseases and schistosomiasis it has been shown to coincide tolerably well with delayed hypersensitivity. However, schistosomiasis is an example of a disease that includes a cytotoxic response in addition to hypersensitivity, the latter minimizing the former (BYRAM & VON LICHTENBERG, 1977). A similar situation will be shown to exist in CL, except that whereas in schistosomiasis the egg is cytotoxic in CL it is the immune mechanisms that are responsible for necrosis. Whatever the cause, necrosis frequently has as a sequel the production of an unorganized granuloma of epithelioid and giant cells, and even if hypersensitivity is perhaps one element in such granulomata, the hypersensitivity may be of any type (LAPORTE,1934). Since the amastigotes of CL are in themselves relatively non-toxic one might have expected that any epithelioid-giant cell granulomata would be the outcome of hypersensitivity and therefore organized, as in tuberculoid leprosy and the L. donovani lesion of Fig. 1. This was found not to be the case. In the whole series of cases observed in this study, threequarters of the granulomata (29/39) were considered to be unorganized and of the post-necrotic type. This was true not only of the early stage (Fig. 2) but of the last stage of development (Fig. 3). Thus the common epithelioid giant cell granuloma of CL is not due directly to ,the parasite, and not due mainly to delayed hypersensitivity, but is a sequel
LEISHMANIASIS
represents
repair of the necrotic
area. Same case as Fig. 2.
to the necrosis. Delayed hypersensitivity is not the main effector mechanism for the elimination of parasites. Distribution
curve
: parasite
index
To find other possible effector mechanisms it was necessary to measure histology against immunological parameters of which the best available was found to be the PI. All the Brazilian mucocutaneous cases had a low PI. But when the CL cases were grouped by their PI they were found to produce a bimodal distribution curve that was a mirror image of the leprosy spectrum (Fig. 4). This held true for
Fig. 4. Distribution CU’WS for the spectrum of leprosy (top) and the parasite index of cutaneous and ~UCOCU~~~~OUS leishmaniasia (bottom).
153
D. S. RIDLEY
both the Ethiopian and the general cutaneous groups. The bimodality of the leprosy curve is due to the instability of patients in the middle of the spectrum (BB), and the rapid transition (without treatment) to LL, where cases accumulate because anergy is ahnost total. In CL it appeared that the PI 2 cases were few because of a rapid transition from 3f to l+. There was evidence of a “watershed” between PI 3 and 4, patients above the line deteriorating without treatment, those below it tending to get better. This corresponded to a somewhat similar watershed in leprosy at the BT position. But why in CL should there be a rapid transition through the PI 2 position? And since parasites are being eliminated? why should the process stop with an accumulation of cases at PI 1 to 0, with delay in the last stage of healing? Necrosis-the
effector
mechanism
When the scores for the various characteristics were plotted against the PI, it was found that the most conspicuous feature of all was the peak score of necrosis at 3+ (Fig. 5). Furthermore, the incidence of necrosis at the 3-t level was 100y0; it was found in O/l0 casesat PI 4 and 5, but 10 out of 10 cases at PI 3. Thereafter the incidence fell back to about 50 y0 at PI 2 to 0. Histologically at PI 4 and 5 there was a massive anergic macrophage granuloma (Fig. 6). At PI 3 organisms were found in one or other of two situations: (i) in macrophages similar to those of Fig. 6 but limited to the subepidermal zone; when necrosis occurred in this situation it resulted in ulceration; (ii) in histiocytic clusters in the dermis, each one of which had a necrotic centre, large or
small (Fig. 7). Here the amastigotes were found mainly near the interface between necrotic centre and histiocytes, either extra-cellularly on the periphery of the necrosis but not in the central area, or in primitive giant cells among the histiocytes (Fig. 8) and to a lesser extent in the histocytes themselves. It was concluded that the necrotic centre represented the death of a mass of parasiteladen macrophages, both host cells and parasites, and the histiocytes and giant cells represented the beginning of the post-necrotic granulomatous response, and early transformation of surviving macrophages. From PI 2 to 0 the necrotic centres resolved and the histiocytic clusters were replaced by somewhat smaller unorganized granulomata. Amastigotes were last seen as a rule in fairly well developed giant cells at the centre of a granuloma (Fig. 9). The cellular infiltrate was heavy. The combined evidence of the histological sequence and the correlation of necrosis with the PI scale left little doubt that it was the necrotizing mechanism that was mainly responsible for the rapid fall from PI 3 to PI 1 in these cases. Correlation
of necrosis with lymphocytes plasma cells
At PI 3 necrosis was found in all biopsies irrespective of lymphocytes and plasma cells, though these were usually fairly numerous. Nevertheless, in the series as a whole there was a statistically significant correlation between necrosis and the score for plasma cells in the Ethiopian group (p
5
4
I -73 aI 2 ? 2 2
I
Fig. 5. Meen scores for the 5 histolorrical characteristics
at different
and
levels of the parasite index. All groupS of leishmaniasis.
154
PATHOGENESIS
Fig. 6. Leishmania
Fig. 7. Histiocytic
OF CUTANEOUS
LEISHMANIASIS
in anergic macrophage granuloma.
cluster with necrotic centre, surrounded by dense
infiltrate
(PI 5). H & E x 1275.
of lymphocytes
and plasma
cells.
(PI 3). H & E
X
135.
155
D. S. RIDLEY
Fig. 8. Amastigotes in primitive
Fig. 9. The last surviving (PI 2). H & E x 535.
giant cell in histiocytic
amastigotes
cluster; also a few on edgeof necrotic centre (right).
in well developed
giant cells at the centre of a small histiocytic
Detail of Fig. 7. H & RX 535.
cluster.
There
was no necrosis.
156
PATHOGENESIS
Fig. IO. Moderately
OF CUTANEOUS
LEISHMANIASIS
well organized granuloma with a well developed Langhans’ giant cell near the centre. (PI 0). H & E ‘: 200.
Fig. 11. Less well organized epithrlioid
cell granuloma
in tax
of PI 4. H (cr E x 2%.
157
D. S. RIDLEY
Fig.
12. bWty
macrophage granuloma
in an Ethiopun
tax
Fig. 13. Basophilic bodies in a macrophage. There were no identifiable dense intiltrate of lymphocytes. H & E x 2000.
with r&tiwly
few lymphocytes.
amastigotes present.
(PI 0). H & E x 735.
The macrophage granuloma
is obscured by a
158
PATHOGENESISOF CUTANEOUSLEISHMANIASIS
was intermediate. Over-all the correlation with plasma cells was slightly more significant than that with lymphocytes. These correlations were due partly to a dearth of cells and absence of necrosis at PI 4 and 5, and for the rest to an association at the PI $+ and 0 levels which was highly significant (Table). Table-Correlation of scores for plasma cells (Ethiopian and Brazilian cases) and lymphocytes (others) with incidence of necrosis Plasma cells: Ethiopia and Brazil Lymphocytes : Other cutaneous 0 2+ 1+ 3f Necrosis (cases)
l/l1
4114
13/16
414
It was concluded that PI 4 and 5+ represented anergy, 3+ provided the optimum level of antigen to induce necrosis, but that at low levels of antigen the continuance of the necrotizing mechanism was dependent on the presence of numerous plasma cells and lymphocytes. These conclusions strongly support the experimental results of BRYCESONet al. (1970), though other workers have been unable to repeat them (MAUEL et al., 1975). It remains to explain the dissociation between hypersensitivity and the protective aspects of healing (BRYCESON et al., 1972; PRESTON& DUMONDE, 1976). The role of organized granulomata Although organized granulomata are not the common form in CL, and their distribution in the PI scale is erratic, there was some evidence that they might have assisted in the reduction of a small parasite load in a few cases: although they were often absent in cases with a low PI, the PI was never high in cases with a high scoring organized granuloma, i.e. with large typical Langhans’ giant cells (Fig. 10). Thus these granulomata might assist at the point where the necrotizing mechanism tended to fail. As against this there were two reasons for thinking that organized granulomata, which probably represent delayed hypersensitivity, might be inconsistent with the necrotizing process: (i) by reducing the antigenic load they would make the necrotizing mechanism more dependent on the presence of numerous lymphocytes and plasma cells; and (ii) there was a statistically significant (p <0.05) inverse association between the incidence of organized granulomata and the combined cell scores for plasma cells and lymphocytes (see also KURBAN et al., 1966). The combination of these two factors ought to be a potent inhibitor of the necrotizing mechanism. In this series of cases, however, there were only three examples of well developed, high scoring organized granulomata (Fig. 10); none produced necrosis. Weakly organized granulomata without giant cells (Fig. 11) might be found in any part of the PI scale and they were not incompatible with necrosis. It could be that plasma cells and
lymphocytes are the primary factor in both the induction of necrosis and the inhibition of well developed organized granulomata, the latter being brought about by endogenous desensitization by antibody, as suggested by WARREN (1972) in schistosomiasis. Lysis of amastigotes by macrophages The foregoing remarks apply predominantly to the general group of CL cases. In the Ethiopian and Brazilian groups the incidence of organized granulomata did not differ much from the other group. But in the Ethiopian group particularly, the incidence of necrosis was much less than in the general group, 30% as against 64% of cases (p
and plasma cells as prime movers A number of workers have suggested that cooperation of humoral antibody with cell-mediated immunity might be essential for the elimination of Leishmania. Lymphocytes and plasma cells have been found to be linked with all the immune mechanisms so far considered, of which they appear to be the prime movers in one way or another. But the attribution of responsibility to one cell type as against the other has been difficult. The numbers piesent in lesions was often large and quantitatively significant. But it often appeared as if the combined score was of more importance than that of each particular cell type. In one case in which a skin biopsy showed a resolving necrotizing lesion with a moderate number of plasma cells, a lymph node biopsy showed a pure lymphocytic occupation of the paracortex, besides other parts of the node. In two cases of CL the lymphocytes in the skin lesion proved to be B cells, the same as in BL leprosy (MARIAN RIDLEY, personal communication): It is in BL leprosy that lymphocytes are quantitatively important in stabilizing immunity in lesions (RIDLEY, 1974). The explanation is not understood.
159
D. S. RIDLEY \ 5
\ \OA \ \
\
A
;\
0
\
\
A \
\
\
\
\ ’
4
\
0
\
A
\
‘\ -----‘T-------
____ \
3
\
A
\
8A8
\
A
A
\---------\ \ A
healing
$,4
0
.
\ \
\
\
\
I Cu
Self
_____
\ H
Cutaneous (Ethiopia) cutoneaJs (other) Mucocutanecur (Brazil)
\ \
2
E 2
\ A
A'\,
A
\ A
\
\
\
\ \
\
\ \
\ \
‘\8
I
i+
8
/+
.w
A
'\\ \
\ \
fpo
\
m&j
=
8j$o
d
0
k,
Q
fi
0
0
0
\ \
0
0
\
fj
‘\y
\ \
0
I
\ \
2 Scae
3 : lymphocytes
Fig. 14. The two dimensional
The spectrum of cutaneous leishmaniasis It appears that the spectrum of CL, if there is a spectrum, is composed of three strands : a necrotizing mechanism, an intracellular lysing mechanism and in a few cases organized granulomata which probably represent delayed hypersensitivity. These mechanisms are set in operation by the co-operation or interaction of cellular immunity and humoral antibody, activated by the stimuli of different species of parasite in hosts of different ethnic origin with varying immune potential (BRADLEY, 1977; GARDENER,
1977).
Alternatively, these immunological mechanisms and the prime moving lymphocytes and plasma cells, measured by their histological scores, can be lumped together to produce a two dimensional spectrum in which the parasite load is represented vertically and the histological scores horizontally (Fig. 14). Viewed in this way, the limited evidence so far available suggests that in the absence of treatment, cases above the line tend to move upwards and to the left, those below the line downwards and to the right. Self-healing is more likely to come about when the right hand margin of the pyramid has been crossed. Whether there is here the basis for a clinically useful spectrum or classification of CL remains to be seen. It is clear that further studies are needed,\ and that more attention must be paid to particular types of disease in particular localities, if possible limited to particular species of Leishmania.
4 + plasma
5 cells
spectrum of cutaneow
6 + granuloma
7 f
\ 8
necrceis
leishmaniasis.
The present investigation will have served its purpose if it provides a framework on which the various forms of disease may be inter-related. Conclusion Using an approximately logarithmic parasite index, 5+ and 4+ represented an anergic non-self healing type of disease. An index of 3f provided the optimum antigen load to set in operation a necrotizing mechanism which frequently destroyed host macrophages as well as parasites. Below l+ the necrotizing mechanism was uncertain and dependent on the presence of many plasma cells and lymphocytes. In a few cases hypersensitivity, represented by organized epithelioid cell granulomata, may have assisted in the reduction of scanty parasites; but at the same time it was probably incompatible with the main effector necrotizing mechanism. Hence the dissociation between hypersensitivity and self-healing. An alternative effector mechanism was seen in a number of Ethiopian and Brazilian cases, by which the macrophages appeared to have lysed the parasites instead of being themselves lysed in the immune process. Acknowledgements In addition to the colleagues whose valued assistance is acknowledged in the text, I am grateful to Dr. A. D. M. Bryceson for several useful comments. Mr. M. J. Goddard kindly undertook the statistical analysis.
160
PATHOGENESIS
OF CUTANEOUS
References
Bjune, R., Barnetson, R. StC., Ridley, D. S. & Kronvall, G. (1976). Lymphocyte transformation tests in leprosy; correlation of the response with inflammation of lesions. Clinical and Experimental Immunology, 25,85-94.
Bradley, D. J. (1977). Regulation of Leishmania populations within the host. II. Genetic control of acute susceptibility of mice to Leishmania donovani infection. Clinical and Experimental Immunology, 30, 130-140. Bradley, D. J. & Kirkley, J. (1977). Regulation of Leishmania populations within the host. I. The variable course of Leishmania donovani infections in mice. Clinical and Exaerimental Immunoloev. U.Z. 30, 119-129. Bryceson, A. D. M., Bray, R. S., Wolstencroft, R. A. & Dumonde. D. C. (1970). Immunitv in cutaneous leishmaniasis of the guinea pig. I
Clinical and Experimental
Immunology, 7,301-341.
Bryceson, A. D. M. (1972). In: Essays on Tropical J. Marshall (Editor). Excerpta Dermatology, Medica Monograph. Amsterdam, pp. 230-241. Bryceson, A. D. M., Preston, I?. M., Bray, R. S. & Dumonde, D. C. (1972). Experimental cutaneous leishmaniasis. Clinical and Experimental Immunology, 10, 305-335.
Byram, J. E. & von Lichtenberg, F. (1977). Altered schistosome granuloma formation in nude mice. American Journal of Hygiene, 26, 944-956.
Tropical
Medicine
and
Destombes, P. (1960). Application de concept de “systematisation polaire” aux leishmanioses cutanees. Bulletin de la Socie’te’ de Pathologie Exotique, 53, 299-301. Epstein, W. L. (1967). Granulomatous hypersensitivity Progress in Allergy, 11, 36-88
LEISHMANIASIS
Epstein, W L. (1977). Granuloma formation in man. Pathobiologv Annual. 7. l-30. Fukuhara, S. & Khngmiiller, G. (1976). Elektronmikroskopische Untersuchungen der Leishmaniasis cutanea. Archives for Dermatological Research, 255, 305-316. Gardener, I?. J. (1977). Taxonomy of the genus Leishmania : a review of nomenclature and classification. Tropical Diseases Bulletin, 74, 1069-1088. Kurban, A. K., Malak, J. A., Farah, F. S. & Chaglassian, H. T. (1966). Histopathology of cutaneous leishmaniasis. Archives of Dermatology, 93, 396-401. Laporte, R. (1934). Histologie des reactions locales d’hypersensibilite chez le cobaye (reactions allergiques a la tuberculine et reactions anaphylactiques). Annales de I’lnstitut Pasteur, 53,598-640. Mauel, J., Behin, R., Biroum-Noerjasim & Rowe, D. S. (1975). Mechanism of protective immunity in experimental cutaneous leishmaniasis of the guinea pig. I. Lack of effects of immune lymphocytes and of activated macrophages. Clinical and Experimental
Immunology,
20, 339-350.
Preston, I?. M. & Dumonde, D. C. (1976). In: Immunology of parasitic infections. S. Cohen & E. H. Sadun (Editors). Oxford, pp. 167-202. Ridley, D. S. (1974). Histological classification and the immunological spectrum of leprosy. Bulletin of the World Health Organization,
51, 451-465.
Turk, J. L. & Bryceson, A. D. M. (1971). Immunological phenomena in leprosy and related diseases. Advances in Immunology,
13, 209-266.
Warren, K. S. (1972). The immunopathogenesis of schistosomiasis : a multidisciplinary approach. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 417-432.
ROYAL
OP TROPICAL MEDICINE AND HYGIENE, VOL. 73, No. 2, 1979
SOCIETY
OF TROI’ICAI.
Ordinary
MEDICINE
Meeting
Manson House, Thursday,
AND HYGIENE
’
18th May, 1978
The President: DR. S. G. BROWNE in the Chair
The pathogenesis
of cutaneous
leishmaniasis
D. S., RIDLEY
Hospital for Tropical Diseases,London NW1 OPE The object of the present study is to have a look at the histological spectrum of cutaneous leishmaniasis (CL) in the hope that it might throw some light on the immunological mechanisms at work. The ultimate object would be to produce a classification that indicated prognosis, though this has not yet been achieved. The snectrum of CL, which ranges from the anergic to-the ultra-hypersensitive (;ecidiva) forms, has been compared-to the spectrum of lenrosv (DESTOMBES. 1960; TURK & BRYCESON, 19711. The-difference is that h CL it is the middle of the spectrum that produces the self-healing forms of disease, the two extremes both being non-selfhealing (BRYCESON, 1972). The inability of many patients to cure themselves without long delay, after having successfully eliminated parasites to the point where they are barely detectable, is perplexing. In the present paper, therefore, an attempt is made to relate pathology to immune mechanisms, and to compare -tie results with those in leprosy. The two diseases are found to be fundamentally different despite their many points in common. -
Patients
and methods
The varieties of CL are due not only to differences in immunological response but to species of parasite. To obtain an over-all view of the subject it was necessary to have biopsies from widespread sources. The present results relate mainly to a group of 33 cases of the purely cutaneous form of leishmaniasis seen at the Hospital for Tropical Diseases which represent all the main endemic areas of the world except for two special groups which were considered separately: (i) biopsies from 27 cases from Ethiopia, most of them sent bv Dr. R. StC. Barnetson, and (ii) biopsies from 22 mucocutaneous cases -from Brazil sent to me bv Dr. A. C. Barretto. Dr. C. C. Cuba and Dr. P. D. Marsden. I am most grateful to all these colleagues, including those in London, not only for the biopsies but for essential clinical data. All cases were active at the time of biopsy. In addition to routine histology, various histological characteristics were assessed quantitatively in a manner similar to that used previously in leprosy (BJUNE et al., 1976). These characteristics were numbers of lymphocytes and plasma cells, the development of granulomata both organised and unorganized, and necrosis. The scores were correlated with the parasite load, which was assessed on an approximately logarithmic parasite index (PI).
For an average sized lesion (approximately 2 x 10 objective fields), the scale was as follows: 5 + = ;;stytimerous amastigotes throughout the 4+
= numerous amastigotes in most parts of the lesion 3+ = numerous in a small part of the lesion (> 300 organisms) 2f = single focus of over 30 organisms l+ = single focus of under 30 organisms & = detected in smear, not biopsy -= not detectable in smear. An important practical point was the unreliability of biopsies of ulcers that did not include either the floor or the margin of the ulcer, though reluctance to take such biopsies was understandable. Most biopsies were good, a few were rejected. A well stained, fairly dark, haematoxylin-eosin was found to be the most satisfactory routine stain, better than Giemsa. Other stains were useful in selected cases.
The essential difference from leprosy The histological spectrum of leprosy (RIDLEY, 1974) depends on a number of features, but there is one that can be relied upon in every case. The character of the granuloma from macrophage to epithelioid and giant cell is invariably characteristic of the position in the spectrum. In CL it was found not to be so. When organisms were numerous the cell type nearly always was a macrophage, but when organisms were scanty the character of the granuloma was unpredictable; often there was none present. The granuloma is not the primary histological feature of CL.
Granulomata
(organized and unorganized)
It is necessary to digress to consider another important aspect of the granulomata of CL. There is only one pathway from macrophage to epithelioid and giant cell and it is the same whether the stimulus is the irritating qualities of the agent or the hypersensitivitv of the patient. The character of the granuloma composed of these same cell types is more varied. EPSTEIN (1967.1977) refers to granulomata as organized or unorganized, and he regards organized granulomata as signifying hypersensitivity. Whether this is always true is not certain, but one can see that hypersensitivity, by producing a strong stimulus operative at some distance from an agent, will help to organize cells in a radius around it. The
151
D. S. RIDLEY
Fig. 1. Organized granuloma suggesting hypersensitivity. L. donovani infection rapidly. (By courtesy of Prof. D. J. Bradley). H & E x 1500.
Fig. 2. Early granuloma
formation
(centre) on edge of necrotic
of liver in strain of mouse that eliminated
area (top left). Cutaneous
leishmtiasis.
parasites relatively
H & E X 215.
152
Fig. 3. More fully H & E Y 235.
PATHOGENESIS
developed
granuloma,
still
unorganized.
OF CUTANEOUS
It probably
small granulomata produced by Leishmania donovani in mouse liver (BRADLEY & KIRKLEY, 1977) is a good example of such organization (Fig. 1). More contentious is Epstein’s view that “granulomatous hypersensitivity” as represented here, is a specific entity of unknown nature. In many mycobacterial diseases and schistosomiasis it has been shown to coincide tolerably well with delayed hypersensitivity. However, schistosomiasis is an example of a disease that includes a cytotoxic response in addition to hypersensitivity, the latter minimizing the former (BYRAM & VON LICHTENBERG, 1977). A similar situation will be shown to exist in CL, except that whereas in schistosomiasis the egg is cytotoxic in CL it is the immune mechanisms that are responsible for necrosis. Whatever the cause, necrosis frequently has as a sequel the production of an unorganized granuloma of epithelioid and giant cells, and even if hypersensitivity is perhaps one element in such granulomata, the hypersensitivity may be of any type (LAPORTE,1934). Since the amastigotes of CL are in themselves relatively non-toxic one might have expected that any epithelioid-giant cell granulomata would be the outcome of hypersensitivity and therefore organized, as in tuberculoid leprosy and the L. donovani lesion of Fig. 1. This was found not to be the case. In the whole series of cases observed in this study, threequarters of the granulomata (29/39) were considered to be unorganized and of the post-necrotic type. This was true not only of the early stage (Fig. 2) but of the last stage of development (Fig. 3). Thus the common epithelioid giant cell granuloma of CL is not due directly to ,the parasite, and not due mainly to delayed hypersensitivity, but is a sequel
LEISHMANIASIS
represents
repair of the necrotic
area. Same case as Fig. 2.
to the necrosis. Delayed hypersensitivity is not the main effector mechanism for the elimination of parasites. Distribution
curve
: parasite
index
To find other possible effector mechanisms it was necessary to measure histology against immunological parameters of which the best available was found to be the PI. All the Brazilian mucocutaneous cases had a low PI. But when the CL cases were grouped by their PI they were found to produce a bimodal distribution curve that was a mirror image of the leprosy spectrum (Fig. 4). This held true for
Fig. 4. Distribution CU’WS for the spectrum of leprosy (top) and the parasite index of cutaneous and ~UCOCU~~~~OUS leishmaniasia (bottom).
153
D. S. RIDLEY
both the Ethiopian and the general cutaneous groups. The bimodality of the leprosy curve is due to the instability of patients in the middle of the spectrum (BB), and the rapid transition (without treatment) to LL, where cases accumulate because anergy is ahnost total. In CL it appeared that the PI 2 cases were few because of a rapid transition from 3f to l+. There was evidence of a “watershed” between PI 3 and 4, patients above the line deteriorating without treatment, those below it tending to get better. This corresponded to a somewhat similar watershed in leprosy at the BT position. But why in CL should there be a rapid transition through the PI 2 position? And since parasites are being eliminated? why should the process stop with an accumulation of cases at PI 1 to 0, with delay in the last stage of healing? Necrosis-the
effector
mechanism
When the scores for the various characteristics were plotted against the PI, it was found that the most conspicuous feature of all was the peak score of necrosis at 3+ (Fig. 5). Furthermore, the incidence of necrosis at the 3-t level was 100y0; it was found in O/l0 casesat PI 4 and 5, but 10 out of 10 cases at PI 3. Thereafter the incidence fell back to about 50 y0 at PI 2 to 0. Histologically at PI 4 and 5 there was a massive anergic macrophage granuloma (Fig. 6). At PI 3 organisms were found in one or other of two situations: (i) in macrophages similar to those of Fig. 6 but limited to the subepidermal zone; when necrosis occurred in this situation it resulted in ulceration; (ii) in histiocytic clusters in the dermis, each one of which had a necrotic centre, large or
small (Fig. 7). Here the amastigotes were found mainly near the interface between necrotic centre and histiocytes, either extra-cellularly on the periphery of the necrosis but not in the central area, or in primitive giant cells among the histiocytes (Fig. 8) and to a lesser extent in the histocytes themselves. It was concluded that the necrotic centre represented the death of a mass of parasiteladen macrophages, both host cells and parasites, and the histiocytes and giant cells represented the beginning of the post-necrotic granulomatous response, and early transformation of surviving macrophages. From PI 2 to 0 the necrotic centres resolved and the histiocytic clusters were replaced by somewhat smaller unorganized granulomata. Amastigotes were last seen as a rule in fairly well developed giant cells at the centre of a granuloma (Fig. 9). The cellular infiltrate was heavy. The combined evidence of the histological sequence and the correlation of necrosis with the PI scale left little doubt that it was the necrotizing mechanism that was mainly responsible for the rapid fall from PI 3 to PI 1 in these cases. Correlation
of necrosis with lymphocytes plasma cells
At PI 3 necrosis was found in all biopsies irrespective of lymphocytes and plasma cells, though these were usually fairly numerous. Nevertheless, in the series as a whole there was a statistically significant correlation between necrosis and the score for plasma cells in the Ethiopian group (p
5
4
I -73 aI 2 ? 2 2
I
Fig. 5. Meen scores for the 5 histolorrical characteristics
at different
and
levels of the parasite index. All groupS of leishmaniasis.
154
PATHOGENESIS
Fig. 6. Leishmania
Fig. 7. Histiocytic
OF CUTANEOUS
LEISHMANIASIS
in anergic macrophage granuloma.
cluster with necrotic centre, surrounded by dense
infiltrate
(PI 5). H & E x 1275.
of lymphocytes
and plasma
cells.
(PI 3). H & E
X
135.
155
D. S. RIDLEY
Fig. 8. Amastigotes in primitive
Fig. 9. The last surviving (PI 2). H & E x 535.
giant cell in histiocytic
amastigotes
cluster; also a few on edgeof necrotic centre (right).
in well developed
giant cells at the centre of a small histiocytic
Detail of Fig. 7. H & RX 535.
cluster.
There
was no necrosis.
156
PATHOGENESIS
Fig. IO. Moderately
OF CUTANEOUS
LEISHMANIASIS
well organized granuloma with a well developed Langhans’ giant cell near the centre. (PI 0). H & E ‘: 200.
Fig. 11. Less well organized epithrlioid
cell granuloma
in tax
of PI 4. H (cr E x 2%.
157
D. S. RIDLEY
Fig.
12. bWty
macrophage granuloma
in an Ethiopun
tax
Fig. 13. Basophilic bodies in a macrophage. There were no identifiable dense intiltrate of lymphocytes. H & E x 2000.
with r&tiwly
few lymphocytes.
amastigotes present.
(PI 0). H & E x 735.
The macrophage granuloma
is obscured by a
158
PATHOGENESISOF CUTANEOUSLEISHMANIASIS
was intermediate. Over-all the correlation with plasma cells was slightly more significant than that with lymphocytes. These correlations were due partly to a dearth of cells and absence of necrosis at PI 4 and 5, and for the rest to an association at the PI $+ and 0 levels which was highly significant (Table). Table-Correlation of scores for plasma cells (Ethiopian and Brazilian cases) and lymphocytes (others) with incidence of necrosis Plasma cells: Ethiopia and Brazil Lymphocytes : Other cutaneous 0 2+ 1+ 3f Necrosis (cases)
l/l1
4114
13/16
414
It was concluded that PI 4 and 5+ represented anergy, 3+ provided the optimum level of antigen to induce necrosis, but that at low levels of antigen the continuance of the necrotizing mechanism was dependent on the presence of numerous plasma cells and lymphocytes. These conclusions strongly support the experimental results of BRYCESONet al. (1970), though other workers have been unable to repeat them (MAUEL et al., 1975). It remains to explain the dissociation between hypersensitivity and the protective aspects of healing (BRYCESON et al., 1972; PRESTON& DUMONDE, 1976). The role of organized granulomata Although organized granulomata are not the common form in CL, and their distribution in the PI scale is erratic, there was some evidence that they might have assisted in the reduction of a small parasite load in a few cases: although they were often absent in cases with a low PI, the PI was never high in cases with a high scoring organized granuloma, i.e. with large typical Langhans’ giant cells (Fig. 10). Thus these granulomata might assist at the point where the necrotizing mechanism tended to fail. As against this there were two reasons for thinking that organized granulomata, which probably represent delayed hypersensitivity, might be inconsistent with the necrotizing process: (i) by reducing the antigenic load they would make the necrotizing mechanism more dependent on the presence of numerous lymphocytes and plasma cells; and (ii) there was a statistically significant (p <0.05) inverse association between the incidence of organized granulomata and the combined cell scores for plasma cells and lymphocytes (see also KURBAN et al., 1966). The combination of these two factors ought to be a potent inhibitor of the necrotizing mechanism. In this series of cases, however, there were only three examples of well developed, high scoring organized granulomata (Fig. 10); none produced necrosis. Weakly organized granulomata without giant cells (Fig. 11) might be found in any part of the PI scale and they were not incompatible with necrosis. It could be that plasma cells and
lymphocytes are the primary factor in both the induction of necrosis and the inhibition of well developed organized granulomata, the latter being brought about by endogenous desensitization by antibody, as suggested by WARREN (1972) in schistosomiasis. Lysis of amastigotes by macrophages The foregoing remarks apply predominantly to the general group of CL cases. In the Ethiopian and Brazilian groups the incidence of organized granulomata did not differ much from the other group. But in the Ethiopian group particularly, the incidence of necrosis was much less than in the general group, 30% as against 64% of cases (p
and plasma cells as prime movers A number of workers have suggested that cooperation of humoral antibody with cell-mediated immunity might be essential for the elimination of Leishmania. Lymphocytes and plasma cells have been found to be linked with all the immune mechanisms so far considered, of which they appear to be the prime movers in one way or another. But the attribution of responsibility to one cell type as against the other has been difficult. The numbers piesent in lesions was often large and quantitatively significant. But it often appeared as if the combined score was of more importance than that of each particular cell type. In one case in which a skin biopsy showed a resolving necrotizing lesion with a moderate number of plasma cells, a lymph node biopsy showed a pure lymphocytic occupation of the paracortex, besides other parts of the node. In two cases of CL the lymphocytes in the skin lesion proved to be B cells, the same as in BL leprosy (MARIAN RIDLEY, personal communication): It is in BL leprosy that lymphocytes are quantitatively important in stabilizing immunity in lesions (RIDLEY, 1974). The explanation is not understood.
159
D. S. RIDLEY \ 5
\ \OA \ \
\
A
;\
0
\
\
A \
\
\
\
\ ’
4
\
0
\
A
\
‘\ -----‘T-------
____ \
3
\
A
\
8A8
\
A
A
\---------\ \ A
healing
$,4
0
.
\ \
\
\
\
I Cu
Self
_____
\ H
Cutaneous (Ethiopia) cutoneaJs (other) Mucocutanecur (Brazil)
\ \
2
E 2
\ A
A'\,
A
\ A
\
\
\
\ \
\
\ \
\ \
‘\8
I
i+
8
/+
.w
A
'\\ \
\ \
fpo
\
m&j
=
8j$o
d
0
k,
Q
fi
0
0
0
\ \
0
0
\
fj
‘\y
\ \
0
I
\ \
2 Scae
3 : lymphocytes
Fig. 14. The two dimensional
The spectrum of cutaneous leishmaniasis It appears that the spectrum of CL, if there is a spectrum, is composed of three strands : a necrotizing mechanism, an intracellular lysing mechanism and in a few cases organized granulomata which probably represent delayed hypersensitivity. These mechanisms are set in operation by the co-operation or interaction of cellular immunity and humoral antibody, activated by the stimuli of different species of parasite in hosts of different ethnic origin with varying immune potential (BRADLEY, 1977; GARDENER,
1977).
Alternatively, these immunological mechanisms and the prime moving lymphocytes and plasma cells, measured by their histological scores, can be lumped together to produce a two dimensional spectrum in which the parasite load is represented vertically and the histological scores horizontally (Fig. 14). Viewed in this way, the limited evidence so far available suggests that in the absence of treatment, cases above the line tend to move upwards and to the left, those below the line downwards and to the right. Self-healing is more likely to come about when the right hand margin of the pyramid has been crossed. Whether there is here the basis for a clinically useful spectrum or classification of CL remains to be seen. It is clear that further studies are needed,\ and that more attention must be paid to particular types of disease in particular localities, if possible limited to particular species of Leishmania.
4 + plasma
5 cells
spectrum of cutaneow
6 + granuloma
7 f
\ 8
necrceis
leishmaniasis.
The present investigation will have served its purpose if it provides a framework on which the various forms of disease may be inter-related. Conclusion Using an approximately logarithmic parasite index, 5+ and 4+ represented an anergic non-self healing type of disease. An index of 3f provided the optimum antigen load to set in operation a necrotizing mechanism which frequently destroyed host macrophages as well as parasites. Below l+ the necrotizing mechanism was uncertain and dependent on the presence of many plasma cells and lymphocytes. In a few cases hypersensitivity, represented by organized epithelioid cell granulomata, may have assisted in the reduction of scanty parasites; but at the same time it was probably incompatible with the main effector necrotizing mechanism. Hence the dissociation between hypersensitivity and self-healing. An alternative effector mechanism was seen in a number of Ethiopian and Brazilian cases, by which the macrophages appeared to have lysed the parasites instead of being themselves lysed in the immune process. Acknowledgements In addition to the colleagues whose valued assistance is acknowledged in the text, I am grateful to Dr. A. D. M. Bryceson for several useful comments. Mr. M. J. Goddard kindly undertook the statistical analysis.
160
PATHOGENESIS
OF CUTANEOUS
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