Leishmania tropica: Pathogenicity and in vitro macrophage function in strains of inbred mice

EXPERIMENTAL

PARASTOLOGY

Leishmania

tropica:

48, 81-91 ( 1979)

Pathogenicity and in Vitro Macrophage in Strains of Inbred Mice

Function

REZA BEHIN, JACQUES MATJEL, AND BERNARD SORDAT World Health Organization Immunology and Training Centre, and Department of Cell Biology of the Swiss Institute for Experimental Cancer Research, Chemin des Boveresses, 1066 Epalinges, Switzerland (Accepted for publication

21 March 1979)

BEHIN, R., MAUEL, J., AND SORDAT,B. 1979. Leishmania tropica: Pathogenicity and in vitro Macrophage function in strains of inbred mice. Experimental Parasitology 48, 8191. Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 x 10’ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and GH/He) recovered from the infection and their lesions healed within 3 to 5 months, The other strains, with the possible exception of C57Bl/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/B, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu+). The responses of C57Bl/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con Astimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supematant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin. INDEX DESCRIPTORS: Leishmania tropica major; Leishmania enriettii; Protozoa, parasitic; Mouse, inbred strains; Mouse, nude; Macrophage, activation; Concanavalin A; Toxoplasma gondii; Lymphokine; Syngeneic; Allogeneic; Hypersensitivity, delayed type. INTRODUCTION

nature. Human infection is acquired during travel or working period in territories occupied by the reservoir host. The disease in man is accompanied by early development of delayed type hypersensitivity (DTH) and is characterized by a relatively

~khmania tropica major, the causative of rural cutaneous leishmaniasis of man in the Middle East and South Central Russia, is a zoonotic parasite infecting primmily species of gerbils and jirds in agent

81 0014-4894/79/040081-11$03.00/O All

Copyright @ 1979 rights of reproduction

by Academic Press, Inc. in any form reserved.

82

BEHIN,

MAUEL,

short incubation period, an acute exudative necrotic phase and spontaneous recovery (Adler 1964; Heyneman 1971; Safjanova and Aliev 1973). Inoculation of freshly isolated parasites in the skin of the ear of golden hamster was shown to produce ulcerative lesions and occasional visceralization (Saf’janova and Aliev 1973). Intracutaneous inoculation of the parasite in the shaved skin of CBA mice has been shown to produce DTH, ulceration, self-cure, and resistance to reinfection, therefore resembling cutaneous leishmaniasis of man (Preston 1973; Preston and Dumonde 1976 ) . In a colloquium on the ecology of leishmaniasis which was held in Montpellier, France, in 1974, we described two patterns of infection by Leishmania tropica major in various strains of inbred mice, a healing and a nonhealing pattern, depending on the strain of the host used. We also reported in the same meeting results of some preliminary observations concerning a difference in the efficacy of activated macrophages from different mouse strains to destroy the ingested parasite (Behin et al. 1977). The object of the present report is to show that the course of infection of various inbred mice with L. tropica major is largely host strain dependent and to provide further information on the role of activated macrophages of various mouse strains in dealing with the parasite. lM~~~~~~~

AND METHODS

Host animals and parasites. Three- to five-month-old inbred mice of the following strains: CBA/Ca, DBA/B, &H/He, A/Jax, C57B1/6, Balb/c, Balb/c( nu+), and nude nu/nu of Balb/c origin were obtained from the breeding colony of the Swiss Cancer Institute. Animals of same sex and age were used in each experiment. (Bokhara Leishmania tropica major strain) was routinely maintained in mice in the form of a skin lesion to avoid possible

AND SORDAT

loss of virulence due to prolonged cultivation. Freshly isolated parasites were propagated in blood-agar cultures and the promastigotes were used to inoculate mice intracutaneously on the shaved back just above the base of the tail. Thirty mice in three groups of 10, from as many strains as possible, were injected with 50 d of a parasite suspension containing either 5, 10, or 20 x lo6 washed promastigotes (it must be noted that not more than 25 to 30 d can be put into the skin of the mouse, the rest of the inoculum runs subcutaneously). The longest and the shortest diameter of lesions were measured monthly and results are expressed as the mean of the two measurements. Leishmania enriettii was used as the control index of macrophage activation; we have shown previously (Behin et al. 1975) that this parasite disappears within activated mouse macrophages in 24 to 48 hr. The parasite was maintained in the form of a nasal lesion in the guinea pig. The amastigotes of L. enriettii were used for infecting macrophages and they were prepared from the nasal lesions, as described before (Mauel et al. 1975). Due to difficulty in obtaining the amastigotes of L. tropica from skin ulcers of mice, in noncontaminated form and in large quantity, the promastigotes of the parasite were used for infecting macrophages. Media and chemicals. Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum (DS), was used for culture of peritoneal macrophages. For lymphocyte culture the same medium was further supplemented with 200 mg/liter L-arginine, 36 mg/liter L-asparagine, 800 mg/liter L-glutamine, and 10 mg/liter folic acid ( Fluka AG, Buchs, Switzerland). Concanavalin A (Con A) was purchased from Pharmacia, Uppsala, Sweden, and doublestranded polyinosinic-polycytidylic acid (PolyI:C), from Biochemical Inc., Wisconsin. All solutions were prepared in pyrogenfree triple distilled water.

L.

tTOpiCU:

PATHOGENICITY

Macrophge culture, infection, and activation. Peritoneal macrophages of intact mice of various strains were collected 3 days after intraperitoneal injection of 3 ml sterile 2% (w/v) starch in normal saline. The cells were washed once by centrifugation, resuspended in DS and 2 ml containing 2 to 3 x lo6 cells was distributed in 35mm diameter Falcon plastic petriplates in which 4 round 12-mm sterile cover glasses had been placed. When cell attachment was completed, plates containing macrophages of each mouse strain were then divided into two groups. One group was infected with 7 x lo6 amastigotes of L. enriettii and the other groups with 10 x lo6 promastigotes of L. tropica. After overnight incubation in a humid COe incubator (5% C02, 95% air), plates were washed three times by addition and suction of fresh medium to remove free-floating parasites. Cells were then overlaid with 2 ml DS before activation was attempted. Activation of macrophages was achieved in the following three ways. (a) Lymphocytes were prepared from intact mice of various strains by gentle grinding of a spleen in a glass tissue grinder. The cells were then washed by centrifugation at 300g for 5 min and resuspended in enriched DS. The cell suspension was left to stand at 4 C for 10 min to allow the tissue debris to settle. The top layer was then removed and the viable cells were determined by dye exclusion test and adjusted to 5 x 10E/ml. For activation of macrophages, 1 ml syngeneic lymphocytes and 0.1 ml of Con A (final concentration: 5 pg/ml) were added to appropriate macrophage-containing plates and incubation was resumed. Control cultures received either 1 ml of lymphocytes or Con A alone. (b) The second method of activation was achieved by addition to macrophage cultures of a lymphokine-rich cell-free supematant prepared from allogeneic or syngeneic lymphocytes which were incubated with Con A and occasionally with

AND

MACROPHAGE

FUNCTION

S3

polyI:C for 72 hr. The lymphocytes were prepared as described above and used at 5 x loo viable cells/ml. The concentration of Con A and polyI:C were of 5 and 50 pg/ml, respectively. Supematants prepared in this manner were found to activate macrophages when used at a concentration of 15 to 30% in DS. The media from all macrophage cultures were renewed every 24 hr. Those destined for activation were pulsed with appropriate amount of the supernatant. A lymphokine-rich supernatant was also prepared from spleen lymphocytes of mice chronically infected with Toxoplasma gondii in the presence of toxoplasma antigen and polyI:C. To each milliliter of lymphocytes in enriched DS containing 5-10 x lo6 viable cells, 0.25 ml of a toxoplasma lysate which originally contained 5 x lo6 trophozoites/ml, was added (for details see Behin et al. 1975). PolyI:C was also added at a final concentration of 50 pg/ml. After 72 hr of incubation at 37 C, the mixture was centrifuged at 300 g for 10 min and the supernatant was titrated for potency. (c) The third method of activation consisted of the combination of the first two techniques. Parasitized macrophages were coincubated with normal lymphocytes and Con A as in method (a). After 24 hr, macrophage plates were washed free from lymphocytes and fresh DS with an appropriate amount of lymphokine-rich supernatant was added to macrophage cultures. This latter step was repeated every 24 hr for 3 days. Addition of supernatant was omitted from control cultures. Assessment of results. Twenty-four hours postactivation, one of the four coverslips was removed from each dish, rinsed in PBS, and stained with Giemsa. The number of parasites per 100 macrophages was determined by counting 500 cells with a 100 X immersion oil lens, in several randomly chosen fields on each coverslip. Each experiment was repeated several times for confirmation of observed results.

BEHIN, SXAUEL, AND SORDAT

i

!

.. . : I-l-L: I I

. -

Ulcer

I; -

Scar

2

3

Months

after

infectIon

FIG. 1. Course of infection and size distributions of cutaneous lesions of five inbred strains of mice (10 mice per group) infected with 5 X lo6 promastigotes of Leishmania tropica major. RESULTS

The Course of Leishmania tropica Infection in Various Strains of Mice Inoculation of at least 5 X lo6 actively motile promastigotes of L. tmpica into the shaved skin of various mouse strains used in this study invariably produced a palpable nodule within 1 week. Central ulceration began on the second week, particularly with higher doses of parasites, and grew larger with time. By 1 month large ulcers were present on all infected animals, regardless of their strain. The gross morphology of the lesion in all mouse strains with the exception of that of DBA/B, was a naked, clean, punched-out ulcer without pus formation or marked inflammatory reaction on the margin. The lesion of DBA/S, on the other hand, was exudative, resulting in formation of a crust which remained permanently over the lesion and slowly increased in thickness and diameter. The outcome of the infection, however, differed depending on the strain of animal used. In this regard, all the mouse strains used in this study could be divided into two general categories, the healer and the

nonhealer strains. The first category was comprised of CBA/Ca and C&H/He strains whose infections with as many as 20 x lo6 parasites, were spontaneously cured within 3-5 months. The second category included DBA/2, A/Jax, and to some extent the C57B1/6 strains, whose lesions, due to as few as 5 x lo6 inoculated parasites, never healed, or did so on a very small percentage of animals. The inclusion of C57Bl among nonhealer strains is rather arbitrary and it should perhaps be considered as an intermediate between the healer and nonhealer categories. Three experiments were performed to study systematically the course and evolution of lesions on five inbred strains of mice infected with 5, 10, or 20 x lo8 motile promastigotes of L. tropica major. Ten mice from each strain were used for each dose and the lesion sizes were measured monthly or biweekly for a period of 4-5 months. Figures 1, 2, and 3 depict one of the experiments, in which common variations among individuals within the groups were represented. As shown on the figures, the diameter of lesions of CBA and C&I mice, infected with as many as 20 X 104

L.

g .-z u) .-0’ !

t?‘OpiCU:

PATHOGENICITY

I

20 14 14 17 14 15 14 13 12 11 10

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-Ulcer

MACROPHAGE

044/Z

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1

Months

2

after

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85

FUNCXION

CIA/C,

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infection

I, - Scar

FIG. 2. Course of infection of mice major.

(10 mice

per group)

and size distributions of cutaneous lesions of five inbred strains infected with 10 X 10’ promastigotes of La’shmania troplca

parasites, did not exceed 6 mm and variation among individuals in a group was small. The course of infection of these two strains was also similar; the lesion size reached its maximum 2 months after infection and progressively regressed afterward. The lesion in CBA mice was faster in heal-

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ing, while recovery in certain individuals among C&H mice was delayed up to 5 months and occasionally more. The infections of DBA/2 and A/Jax mice, although morphologically different, followed a similar course, which differed in many respects from that of CBA and CSH animals. The

. A/;,,

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FIG. 3. Course of infection and size distributions of cutaneous lesions of five inbred strains of mice (10 mice per group) infected with 20 X 10” promastigotes of Leishmania tropica

major.

86

BEHIN,

MAVEL,

AND

TABLE

SOBDAT

I

Effect of Activated Mouse Macrophages on the Number of Intracellular Leishmania enriettii and Leishmania tropica Cultures”

CBA/Ca b-1

DBA/2 (h)

GH/He @r)

C57B1/6 (hr)

24

48

24

48

24

48

24

48

M@ + Le. + Ly. M+ + Le. + Ly. + Con A

380 212

434 0

253 58

200 0

152 65

142 3

245 209

242 0

M+ + L.t. + Ly. M6 + L.t. + Ly. + Con A

507 620

800 0

217 94

91 14

420 202

414 301

218 326

324 151

0 M+, Macrophages; Ly., lymphocytes; L.e., Leishmania enriettii, LA., Leishmania tropica. b The values represent numbers of intracellular parasites per 100 macrophages.

sizes of ulcers increased with time, often reaching a diameter of over 16 mm and persisting throughout the life of the animals. It must be noted however that a few individuals among A/ Jax mice, particularly those infected with 5 x 10” parasites, developed small lesions, not exceeding 5 to 6 mm, that eventually healed. The evolution of the lesions in C57B1/6 animals showed the largest variability among individuals within a group. The rate of recovery from infection with 5 x lo6 parasites often reached as high as SO%, but that of animals infected with higher doses did not exceed 50%. A peculiar feature of persisting lesions on C57 mice was the invasiveness of the ulcer which progressively eroded the skin, running down the leg and toward the scrotum. The invasive ulcers were not present on all the animals infected with high doses of the parasite and they were not associated with large numbers of parasites in skin smears. In fact, the number of parasites in smears made from scraping tissue from the border of such lesions was very scanty. Such a phenomenon was rarely seen on A/Jax but not on any other mouse strains so far tested, including on DBA/2 mice in which parasite density in the lesion is enormous. In two smaller scale experiments comprising five animals per group, two other strains of mice plus their hybrid, namely

Balb/c and nu/nu of Balb/c origin, and the Balb/c( nu+), were infected with 5 x lo6 promastigotes of L. tropica. The infection in the three groups and in both experiments followed a nonhealing course. It must be mentioned however that, due to the short life span of athymic nude mice, the final outcome of the infection could never be ascertained, but the highly proliferative nature of the parasite in both skin and viscera of infected nude mice pointed to a fatal outcome. Visceralization invariably occurred in all the infected nude mice and the spleen and liver became hyperplastic with numerous granulomatous foci and massive numbers of parasites. It is interesting to note that the appearance of leishmanial lesions at the site of injection on nude mice was much delayed, as compared with other mice in the same experiment. By the end of the second week postinfection, when an open ulcer was present on Balb/c and Balb/c (nu+), the nudes had only a small nodule. The nodule however started growing rapidly afterward, forming a large protruding abscess. The dead skin formed a crust over the abscess, often preventing it from becoming an open ulcer with secondary infection. The abscess contained yellowish white pus in which numerous motile promastigotes could be found. Apparently lowered temperature due to superficiality of ulcer and

L.

t?@CU:

PATHOGENICITY

AND

TABLE Effect

of Macrophage Intracellular

Expt.

MACROPHAGE

87

FUNCTION

II

Activation by Allogeneic Lymphocytes and Con A on Leishmania enriettii and Leishmania tropica

Cultures”

L. enriettii

(hr)

L. tropica

(hr)

M+

LY.

Con A

24

48

24

48

I

DBA DBA GH GH

CIH C,H DBA DBA

+ +

260b 198 112 120

324 0 294 0

>500 > 500 349 356

>500 >500 337 20

II

DBA DBA CBA CBA

CBA CBA DBA DBA

+ +

325 239 299 220

318 23 301 8

344 321 308 265

363 324 300 20

QM+, Macrophage; Ly., lymphocyte. b The values represent number of intracellular

parasites

the absence of blood vessels in the necrosed tissue caused the transformation of liberated amastigotes to promastigotes. Leishmanicidal Capacities of Activated Mouse Macrophages In preliminary experiments on activation of macrophages of various mouse strains by syngeneic lymphocytes and Con A, it was noticed that while activated macrophages of all mouse strains were effective in destroying ingested Leishmania enriettii within 24 to 43 hr, certain strains were not equally capable of destroying L. tropica even after 96 hr. Table I shows the results of an experiment in which the parasiticidal capacities of activated macrophages of four mouse strains were simultaneously tested, using L. enriettii and L. tropica in parallel. It can be seen that 48 hr after the addition of lymphocytes and Con A, L. enriettii was totally destroyed in macrophages of all four strains, while L. tropica was still persisting. This parasite was only destroyed by macrophages of healer origin (CBA, C,H) after 72 hr. The number of parasites did not change in nonhealer (DBA/2) macrophages by this time, and it was reduced to half in activated C57Bl cells.

per 100 macrophages.

The addition of syngeneic lymphocytes and Con A to CBA macrophages, occasionally failed to sufficiently activate the latter cells to kill L. tropica, although L. erwiettii was killed within similarly activated cells (see Table IV). In a few experiments spontaneous activation occurred in CBA and C57 macrophages causing partial destruction of L. enriettii in the absence of lymphocytes or Con A (see Tables III and V). On no occasion did activation by the above methods result in destruction of L. tropica within DBA/2 macrophages, even after 120 hr. However, the killing of L. tropicn was occasionally observed in activated macrophages of A/Jax mouse which is a nonhealer strain. Activation Allogeneic

of Macrophages by Con A and Mouse Lymphocytes

Experiments were designed to determine whether failure of nonhealer macrophages to destroy L. tropica could be attributed to an intrinsic defect of the macrophages themselves, or to an incapacity of nonhealer lymphocytes to induce proper activation. Macrophages of nonhealer origin were thus exposed to ConA-stimulated lymphocytes from healer animals and con-

88

BEHIN,

IMAUEL,

versely, healer macrophages to nonhealer lymphocytes. The effect of such activated cells on the fate of L. tropica was then determined. The destruction of L. enriettii was used as an index of activation. In the control cultures, parasitized macrophages received the same allogeneic lymphocytes but no Con A. Results of two experiments are presented in Table II, which clearly show that activation of macrophages was achieved by allogeneic lymphocytes and Con A, irrespective of the origin of each cell type, as evidenced by destruction of L. enriettii. However, destruction or reduction of the number of L. tropica was only accomplished in activated macrophages of CBA and C3H origin.

M+ M+ M+ M+

+ + + +

L.e. L.e. + supernatant LA. L.t. + supernatant

III

on Intracellular

Leishmania

DBA/S (hr)

CBA/Ca (hr)

by the

Since the presence of normal lymphocytes and Con A or sensitized lymphocytes and toxoplasma antigen seemed to induce greater level of activation but tended to cause the culture condition to deteriorate, a combination of the first two methods was attempted with satisfactory results. Twentyfour hours after incubation of parasitized macrophages with normal lymphocytes and ConA or toxoplasma-sensitized lymphocytes and the antigen, the cultures were washed free from lymphocytes and supplemented with fresh media containing a small amount of the supernatant. The washing and continuous pulsing of the appropriate plates with the supernatant prolonged the life of the cultures and resulted in a condition of sustained activation. Table IV shows the results of an experiment in which macrophages of four mouse strains were activated by normal lymphocytes and Con A, and Table V shows the results of another experiment in which macrophages of five mouse strains were activated by sensitized lymphocytes and toxoplasma antigen. As can be seen from

TABLE

Culturesa

within 24 to 48 hr however, the level always sufficient to tropica by 72 hr or

Activation of Macrophages Combined Method

Addition to cultures of parasitized macrophages of an active cell-free supernatant which was prepared by incubation of normal lymphocytes and Con A, or sensitized lymphocytes and toxoplasma antigen, invariably induced activation of macrophages of all mouse strains tested. The technique had the advantage of requiring no further addition of lymphocytes. Therefore, the macrophage cultures could be washed and pulsed with a small amount of the supernatant every 24 hr. Although the longevity of cultures was improved and activation was quite evident by destruction

Macrophages

SOBDAT

of ingested L. enriettii as shown in Table III, of activation was not cause destruction of L. longer.

Activation of Macrophages by Lymphokine-rich Supernutants

Effect of Supernatant-Activated

AND

enriettii

C3H/He (hr)

and Leishmania C57B1/6 (hr)

tropica A/Jax (hr)

24

72

24

72

24

72

24

72

24

72

186b 28 250 214

104 0 322 0.6

326 299 139 113

296 0 237 50

123 137 295 278

194 88 437 457

291 45 102 19

8 0 297 0

220 127 215 139

203 0 228 3

a M+, Macrophage; L.e., I,. enriettii; L.t., L. tropica. * The values represent numbers of intracellular parasites

per 100 macrophages

L.

tTOfIliCU:

PATHOGENICITY

AND

TABLE

MACROPHAGE

89

FUNCTION

IV

Effect on Leishmania enriettii and Leishmnia tropica of Mouse Macrophages Activated Initially by Con A-Stimulated Lymphocytes then by Lymphokine-rich Supernatant Cultures”

CBA (hr)

DBA (hr)

24

72

24

72

24

72

24

72

188”

262

110

112

156

168

137

195

Supnt M+ + L.t. M+ + L.t. + Ly. + Con A

85

196

118

172

98

142

71

154

M+ + Lt.+ r1LY + Con A

;:

‘:.6

1::

M+ + L.e. M+ + L.e. + Ly. + Con A

‘1.4

C,H (hr)

1:;

“1.4

C57 (hr)

1::

::

Supnt. a M+, Macrophage; Ly, lymphocyte; Supnt, supernatant; L.e., L. enriettii; L.t., L. tropica. b The values represent numbers of intracellular parasites per 100 macrophages.

respond differently to infection with L. major. Although all the strains tested were found susceptible to the infection, the fate of the infection, however, differed among strains. WhiIe the CBA/Ca and C3H/He mice were capable of destroying the parasite and self-healing in 3 to 5 months, the DBA/2, A/Jax, Balb/c, Balb/c (nu+), and Balb/c (nu/nu) aniDISCUSSION mals developed very large ulcers and reDifferences in susceptibility of various mained infected throughout their lives. mouse strains to infection with a leish- Somewhat similar findings have also been mania parasite was first described by Brad- cited by Preston and Dumonde (1976). ley (1974) and Bradley and Kirkley The observed difference in responses of (1977a, b) who, among some 25 strains of inbred mice to infection with L. tropica inbred mice infected with Leishmania indicates a genetic basis for protective imdonovani of Ethiopian origin, found cer- mune responses of healer animals and the tain strains acutely susceptible and some failure of nonhealer mice to resolve the strains acutely resistant to the infection. infection. On the other hand, the fulminatThe susceptible strains were further sub- ing infection of athymic nude mice points divided into those that allowed uncon- to thymus-dependency of the healing protroled proliferation of the parasite and cess. This is in concordance with the findthose that, after a period of patency and ing of Preston et al. (1972), who showed partial proliferation of the parasite, that thymectomized, irradiated CBA mice mounted an immune response and signifi- (a healer strain) often failed to resolve cantly reduced the number of parasites in their infection even after 20 weeks. It foltheir livers. A rather similar situation is lows, therefore, that the resistance of healer evident in the present study in which dif- mice and susceptibility of nonhealer aniferent strains of inbred mice were found to mals are manifested through immunologi-

Tables IV and V, under optimal cultural condition and sustained activation, which was achieved by the combined method, activated macrophages of all five strains, whether healer or nonhealer, were equally capable of destroying or significantly reducing the numbers of ingested L. enriettii and L. tropica in 48 to 72 hr.

trapica

90

BEHIN,

MAUEL,

cal events that take place when infection is initiated or thereafter. The question arises then as how could the nonhealing phenomenon of certain mouse strains with intact functional thymus be interpreted in immunological terms? Killing of L. tropica inside macrophages of healer mice which were activated by lymphocytes of nonhealer animals clearly points to a functional T-cell population in nonhealer mice. On the other hand, destruction of the parasite in macrophages of nonhealer mice in vitro by maintaining a continuous level of activation, also points to potential capacity of these cells to eliminate the parasite from their cytoplasm. Admittedly, the reactivity of “nonhealer” lymphocytes to Con A does not necessarily mean reactivity to leishmania antigen. There might also be a specific defect in recognition and reactivity to leishmania antigen by nonhealer macrophages. However, recent results from our center do not seem to support these notions. It has been found that (a) leishmania-sensitized lymphocytes of nonhealer mice, when incubated with normal syngeneic macrophages that have engulfed L. enriettii, became stimulated and caused activation and, as

AND

SORDAT

the consequence, destruction of the parasite inside macrophages (Mauel et al., unpublished) and (b) lymphocytes from regional lymph nodes of nonhealer mice immunized with L. tropica strongly reacted in vitro to the parasite, as shown by a significant increase in the uptake of [3H]thymidine (Louis et al., in preparation). It is evident from these observations that, so far as the in vitro situation is concerned, macrophages of nonhealer mice can present leishmania antigens and lymphocytes of nonhealer mice recognize and react to these antigens as do cells of healer animals. It is conceivable that these potentialities are rendered nonfunctional in vivo, because of some as yet undetermined mechanisms or factors. Preston and Dumonde (1976) have shown that the magnitude of the delayed hypersensitivity reaction is inversely proportional to the infecting doses of L. tropica in CBA mice. Therefore, increased antigenic load seems to coincide with diminished cell-mediated reactivity. We have often been impressed by a difference in the number of parasites present in the lesions of DBA and CBA mice which had received a similar infective dose of L. tropica. While scraping of the lesion of

TABLE

V

Effect on Leishmania enriettii and Leishmania tropica of Mouse Macrophages Activated Toxoplasma gondii Sensitized Lymphocytes and T. gondii Antigen then by Lymphokine-rich CultureP

DBS/2 (hr)

CBA/Ca (hr) 24

w#J + L.e.

72

24

72

b

314~ + L.e.t r]SL. + T.Ag

::

',"

1::

":.4

Initially by Supernatant.

C3H/He (hr)

C57B1/6 (hr)

A/Jax (hr)

24

72

24

72

24

72

137

120

144

161

206

252

152

8

131

0

239

0

Supnt. hlf$ + L.t.

Supnt.. 0 M@, Macrophage; SL., sensitized lymphocytes; T.g.Ag, Toxoplasma gondii antigen; L.t., L. tropica; Supnt., supernatant. 1,The values represent number of intracellular parasites per 100 macrophages.

I,. e., L. enriettii;

L. trOpiC@: PATHOGENICITY AND MACROPHAGE FUNCTION

nonhealer DBA animals reveals numerous parasites on microscopic examination, parasites on similar preparations from CBA are often scanty and sometimes hard to Snd. There is also no doubt that intact macrophages of DBA mice provide a better “home” for multiplication of the parasite than do CBA or C3H macrophages (personal observation). It is possible, therefore, that the more hospitable microenvironment of DBA macrophages allows the antigenic load to arrive at the threshold of inhibiting an effective cellular immune response. However, such explanation may not hold true for other nonhealer mouse strains such as A/Jax, since we have no evidence of a greater number of parasites in their lesions or in their macrophages. Results of Con A activation (Table I), a process independent of antigen recognition by lymphocytes or presentation by macrophages, indicate a deficiency in the capacity of “nonhealer” macrophages to decompared to macrophages stroy L. tropica, from healer animals activated under the same conditions. This, together with the results demonstrating that continuous activation of “nonhealer” macrophages eventually induces these cells to destroy the parasite (Tables IV and V) strongly suggests that the threshold of effective activation (with respect to destruction of L. tropica) is higher in “nonhealer” than in “healer” macrophages. It is possible that this “defect,” combined with a possible decreased lymphocyte reactivity in nonhealer animals, is the basis of the mechanisms leading to failure of healing in these hosts. ACKNOWLEDGMENT This work was supported by the U.S. National Institutes of Health through Grant 5ROl AI 13126-02.

REFERENCES ADLER, S. 1964. Leishmunia. In “Advances in Parasitology” ( B. Dawes, ed. ), Vol. 2, p. 3596. Academic Press, London/New York.

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