REVIEWS OF INFECTIOUS DISEASES. VOL. 5, NO.5.

SEPTEMBER-OCTOBER 1983
© 1983 by The University of Chicago. All rights reserved. 0162-0886/83/0505-0008$02.00

The Immunobiology of Leishmaniasis

Richard D. Pearson, David A. Wheeler, From the Divisions of Geographic Medicine and of
Lee H. Harrison, and H. David Kay Rheumatology, Department of Internal Medicine, University
of Virginia School of Medicine, Charlottesville, Virginia

Members of the genus Leishmania are important intracellular pathogens that produce
either cutaneous, mucocutaneous, or visceral disease in many areas of the world. In hu-
mans as well as in other mammals, the parasite is inoculated through the skin as a
flagellated, extracellular promastigote by its arthropod vector, the sandfly. Once in its
mammalian host, the promastigote converts to its amastigote stage, which lacks an ex-
teriorized flagellum and is found solely within mononuclear phagocytes during estab-
lished infection. In vitro, human monocyte-derived macrophages and peritoneal mac-
rophages from several species of rodents can ingest both promastigotes and amas-
tigotes, and they can permit intracellular multiplication of amastigotes only. Although
serum factors may play a role in the pathogenesis of the disease and in protection
against reinfection, the resolution of leishmaniasis is dependent primarily on cell-
mediated immune responses. There appears to be a complicated interplay between cell-
mediated helper and suppressor activities. The outcome of infection in each type of
leishmaniasis depends on the complex and intriguing interaction of virulence factors

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inherent in the parasite and genetically determined host defense mechanisms.

Leishmania are intracellular protozoal parasites
that are responsible for significant morbidity and
mortality in many areas throughout the world.
Leishmaniasis is the general name given to infec-
tion caused by any member of the genus Leish-
mania. Clinical manifestations range from visceral
disease to one of several forms of cutaneous dis-
ease, depending on the infecting species. So seri-
ous is the threat of leishmaniasis in endemic areas
that the World Health Organization has selected it
as one of the six major diseases for study in its
Special Programme for Research and Training in
Tropical Diseases. As a result of this emphasis and
recent interest in leishmaniasis, important ad-
vances have been made toward understanding the
Received for publication June 20, 1982, and in revised form
February 22, 1983.
This work was supported by grant no. 1 ROI AI 184802-01
from the National Institutes of Health and by grant no. RF
79062 from The Rockefeller Foundation.
We are indebted to Dr. Sydney S. Breese, Associate Director
of the Central Electron Microscope Facility at the University of
Virginia School of Medicine, for assistance in obtaining elec-
tron micrographs. We are especially grateful to Ms. Anne
Groschel and Ms. Susan Davis for their help in preparing the
manuscript.
Please address requests for reprints to Dr. Richard D. Pear-
son, Division of Geographic Medicine, Box 485, University of
Virginia School of Medicine, Charlottesville, Virginia 22908.
parasite and defining critical aspects of its interac-
tion with mammalian host defense mechanisms.
The Parasite
Leishmania are dimorphic protozoa, i.e., their life
cycle involves existence in two distinct forms. In
infected mammalian hosts, the parasite is found
almost exclusively within cells of reticuloendothe-
lial origin in its amastigote form, which is 2-3 urn
in length and oval or round in shape and lacks an
exteriorized flagellum (figure 1). In sandfly vec-
tors (Phlebotomus species and Lutzomyia species)
on the other hand, the parasite converts to and is
then transmitted as a flagellated, extracellular
promastigote. Promastigotes have pear- or spindle-
shaped bodies, are 10-15 f.Lm in length and 1.5-3.5
f.Lm in width, and have long unitary flagella (figure
2).
Although minor ultrastructural differences have
been described [1-7], most investigators find it
difficult, if not impossible, to differentiate species
of Leishmania that cause disease in humans, from
one another on the basis of morphology in either
the amastigote or promastigote stages. Determina-
tion of species type was originally based on a vari-
ety of factors such as: (1) the parasite's behavior
in humans; (2) epidemiologic differences asso-
ciated with its geographic distribution; (3) the in-

907
908 Pearson et al.

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Figure 1. (A) Leishmania donovani amastigotes (arrows) are seen in a touch preparation made from an infected
Syrian hamster spleen (Wright-Giemsa stain). Amastigotes are 2-3 J.tm in diameter and have an eccentrically located
nucleus and a dense-staining rod-shaped kinetoplast. The nuclei (N) of host mononuclear phagocytes are also seen.
The bar represents 10 J.tm. (B) Transmission electron micrograph of L. donovani amastigotes isolated from an infected
hamster spleen showing the parasite nucleus (N), flagellum (F), and kinetoplast (K). A dividing parasite is present.
The bar represents 1 J.tm.

volvement of different animal reservoirs; and (4)
transmission by different species of sandflies.
These traditional approaches to speciation have
proved inadequate. Clinical syndromes overlap
and the manifestations of disease can vary, de-
pending in some instances on the mammalian host
that is infected. For example, species of Leish-
mania that produce cutaneous disease in humans
can cause visceral disease in rodents [8]. Because
of these difficulties, several biochemical methods
Immunobiology of Leishmaniasis
Figure 2. Scanning electron micro-
graph of a Leishmania donovani pro-
mastigote (P) being ingested by a hu-
man mononuclear phagocyte (M) in
vitro. Most of the parasite's flagellum
lies within a phagocytic pseudopod
(arrow). The bar represents 5 (.Am.
of speciation have been proposed: isoenzyme de-
termination [9-13], buoyant density analysis of
nuclear and kinetoplast DNA [10, 13, 14], radio-
respirometry [15], and analysis of antigenic differ-
ences in promastigotes or in their excreted factors
[13, 16, 17]. While none of these procedures has
proved completely reliable in and of itself, deter-
mination of isoenzyme patterns is currently the
most widely used method of speciation. The devel-
opment of species-specific monoclonal antibodies
[18] or characterization by restriction endonuc-
lease digestion of kinetoplast DNA may ultimately
provide a superior basis for species identification
and is currently an extremely active area of re-
search.
Clinical Manifestations
The clinical syndromes caused by species of Leish-
mania will be briefly outlined in order to provide
a basis for discussion of the host-parasite interac-
tion. Comprehensive reviews of the epidemiology
and clinical features of leishmaniasis can be found
elsewhere [19-28]. As alluded to earlier, various
species of Leishmania produce either visceral or
one of several forms of cutaneous disease. The
distinct tissue tropism of the various leishmanial
species is poorly understood at present. Sensitivity
to temperature, for example, may be responsible
909
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for limiting the spread of some species to the skin
and mucous membranes.
Visceral Leishmaniasis
In visceral leishmaniasis (Leishmania donovani
complex), amastigotes multiply within mononu-
clear phagocytes throughout the reticuloendotheli-
al system, including the spleen, liver, lymph
nodes, and bone marrow [29]. Intermittent fever,
anemia, and pronounced hepatosplenomegaly are
common. There is usually progressive weight loss,
cachexia, and malaise followed by bacterial super-
infection, a complication that can result in death.
Asymptomatic infections may occur with L. dono-
vani, but the data regarding this question certainly
are not complete [26]. In general, once the stage of
hepatosplenomegaly is reached, host defense
mechanisms are not able to prevent further para-
site multiplication.
Although patients with visceral leishmaniasis
fail to mount delayed-type skin hypersensitivity
reactions to leishmanial antigen (as measured by
the Montenegro test), they do have markedly ele-
vated globulin levels and produce Leishmania-
specific antibodies. Globulin levels as high as 5
g/ 100 ml are occasionally observed, but much of
this globulin is not Leishmania specific [23]. After
successful treatment with a pentavalent organic
910
antimonial agent (e.g., antimony sodium stiboglu-
conate) or an alternative drug, delayed hypersensi-
tivity to leishmanial antigen usually develops
within several months, antibody levels gradually
decline, and there is usually resistance to reinfec-
tion [20]. This series of events has led to specula-
tion that potentially effective cell-mediated host
defense mechanisms are suppressed during active
visceral leishmaniasis.
A condition known as post-kala-azar dermal
leishmaniasis is sometimes seen as a sequel to
treated L. donovani infection. In this syndrome,
widely disseminated cutaneous lesions, which con-
tain L. donovani amastigotes in macrophages,
develop months to years after successful treatment
of visceral leishmaniasis.
Cutaneous Leishmaniasis
The cutaneous forms of leishmaniasis occur in
both the Old World (e.g., Leishmania tropica
complex) and the New World (e.g., Leishmania
mexicana complex and Leishmania braziliensis
complex). Once inoculated by the sandfly into the
skin, it seems apparent that parasites multiply in
macrophages. In classic localized cutaneous leish-
maniasis, a papule forms and eventually ul-
cerates. Lesions usually heal spontaneously within
several months, but a flat, atrophic scar remains
as evidence of the disease. The Montenegro skin
test usually becomes positive four to six weeks
after onset of the lesion. Leishmaniasis recidivans
is a variant of cutaneous leishmaniasis in which
Pearson et al.
isolated or grouped tubercules appear over or
around scars of healed cutaneous ulcers [30].
These new lesions are associated with the persis-
tence of a few parasites in scars or proximallym-
phatic channels.
In diffuse cutaneous leishmaniasis, a relatively
uncommon syndrome, disease starts as a localized
lesion, usually a papule, that does not ulcerate.
Satellite lesions subsequently develop around the
initial papule, and organisms may metastasize to
distant areas of the skin, often to the face and ex-
tremities. The disease runs a protracted course,
but visceral dissemination does not occur. Diffuse
cutaneous leishmaniasis has been described in
both the New World [31-34] and the Old World
[35]. Antibodies are demonstrable, whereas de-
layed hypersensitivity responses to leishmanial
antigen are absent. It has been hypothesized that
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the disease simply represents poor host resistance,
but it is possible that parasite factors playa role
as well. There is evidence suggesting that, as in vis-
ceral leishmaniasis, cell-mediated immune re-
sponses against the parasite are suppressed during
diffuse cutaneous leishmaniasis [34].
Mucocutaneous leishmaniasis is found predom-
inantly in South America, where L. brasiliensis
complex is usually implicated as the causative
species. Patients initially develop cutaneous le-
sions that can be followed months to years later by
destructive mucosal lesions. Because the most
common sites of mucosal involvement are the
cartilaginous structures of the nose, mouth, and
pharynx, gross deformities of the face can result
Figure 3. Patient with mucocutane-
ous leishmaniasis who has severe in-
volvement of the face and concomi-
tant destruction of the nasal septum.
(Photograph courtesy of Dr. Anasta-
do de Queiroz Sousa, Universidade
Federal do Ceara, Fortaleza, Brazil).
Immunobiology of Leishmaniasis
(figure 3). The percentage of patients with cutan-
eous lesions who subsequently go on to develop
mucocutaneous disease is unknown. Isolation of
the organism by direct smear or culture of muco-
sal lesions is difficult. The Montenegro skin test is
positive in most cases, a fact indicating that the
host is capable of mounting cell-mediated immune
responses to leishmanial antigen.
In some respects, the range of histopathological
features in the cutaneous forms of leishmaniasis is
analogous to that of leprosy [19, 27, 33, 36]. At
one end of the spectrum lies diffuse cutaneous
leishmaniasis, in which there is little evidence of
an effective immune response. Vacuolated, heavi-
ly parasitized macrophages are abundant, but
there are few lymphocytes. The cutaneous reac-
tion to leishmanial antigen is negative. The histol-
ogy of diffuse cutaneous leishmaniasis is similar to
that of lepromatous leprosy, in which there is mas-
sive bacterial infection of macrophages and little
cell-mediated immune response. At the other end
of the spectrum lies mucocutaneous leishmaniasis.
In the mucosal lesions, there is a pronounced
mononuclear cell infiltrate, but few if any para-
sites can be found. This situation is analogous to
that of tuberculoid leprosy, in which there is an
intense mononuclear infiltrate with few bacteria
[19, 33].
Treatment of typical cutaneous leishmaniasis
with a pentavalent antimonial drug generally re-
sults in progressive lymphocytic infiltration, a
decrease in parasite density, and reversion to skin
test positivity [27]. Local necrosis of the epidermis
and ulceration may occur. These findings have led
to speculation that antileishmanial therapy re-
duces the parasite burden and permits the expres-
sion of cell-mediated immune mechanisms at
levels that may be sufficient to resolve the disease.
Immune Response to Leishmania
Development of clinical leishmaniasis is depen-
dent on both host and parasite factors. The chal-
lenge is to separate invasiveness, pathogenicity,
and parasite survival into intrinsic host and para-
site components [20]- in other words, to deter-
mine the degree to which the clinical form of leish-
maniasis is an expression of the parasite's genetic
constitution (virulence) or the host's innate suscep-
tibility and capacity to respond (immunity). Al-
though a comprehensive explanation of the im-
911
munobiology of the various forms of leish-
maniasis is not possible, important advances have
been made toward understanding the parasite's
biology and defining critical aspects of their inter-
action with mammalian host defense mechanisms.
These advances are discussed in detail in the sec-
tions that follow. Attention will focus on recent
progress. The older literature is well summaried
elsewhere [19-24, 27] and will be referred to only
as it relates to recent studies.
When a Leishmania-infected sandfly attempts
to take a blood meal from its mammalian host,
promastigotes are inoculated. The precise se-
quence of events that follows inoculation has not
been defined. Although Leishmania exist only
transiently in the promastigote stage, the initial
interaction of promastigotes with mammalian
host defense mechanisms is critical to the develop-
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ment of infection. Based on in vitro studies with
human serum and polymorphonuclear leukocytes,
it appears that promastigotes must escape poten-
tially lethal humoral and cellular host defense
mechanisms in order to gain entrance into macro-
phages. Once Leishmania have become amasti-
gotes within macrophages, resolution of infection
depends predominantly on cell-mediated immune
mechanisms [37-39].
Genetic Control of Resistance to Leishmania
Unfortunately, no data are available about the
'genetic control of susceptibility of humans to
leishmanial infection. On the other hand, it seems
clear that resistance to L. donovani and L. tropica
is genetically determined in mice [40-46]. Some
inbred strains of mice are susceptible to visceral or
cutaneous leishmaniasis, while others are resis-
tant.
Resistance to L. donovani. Bradley and co-
workers have reported that resistance to L. don-
ovani is controlled by a single autosomal gene on
chromosome 1. The gene has been designated Lsh
[41,42]. This genetic locus, which controls natural
resistance to L. donovani in mice, is closely linked
to, and probably identical with, the locus that con-
trols resistance to Salmonella typhimurium [45].
Among susceptible strains of mice, some exhibit
prolonged infection while in others there is a pro-
gressive decrease in parasite burden late in infec-
tion. This late response is controlled by a genetic
locus within or close to the major histocompatibil-
912
ity complex (H-2) [43] as well as by genes asso-
ciated with minor histocompatibility loci (H-11)
and with the Ir-2 locus [44]. Even in susceptible
C57Bl mice, Semprevivo et al. [46] have demon-
strated a spectrum of visceral disease among con-
geneic strains. Unfortunately, the mechanisms(s)
by which resistance to visceral leishmaniasis is
mediated in the various mouse strains has not yet
been defined and may differ from strain to strain.
Resistance to L. tropica. The capacity of cer-
tain murine strains to be infected with L. tropica
is likewise under genetic control [40, 47-53].
BALBI c mice, which are susceptible, have pro-
gressive local disease followed by disseminated in-
fection, while C57Bl, mice which are considered
resistant, can be infected but display localized,
self-healing ulcers. Relative resistance to L. tropi-
ca is controlled by a single autosomal gene dif-
ferent from Lsh and not involved in the H-2 com-
plex. Howard et al. [49] have shown - with radia-
tion chimeras, which are compatible at the H-2
locus - that susceptibility to L. tropica is deter-
mined by the descendants of donor hematopoietic
(bone marrow) cells and not by environmental
factors. The susceptibility of BALB/c mice to
L. tropica appears not to be due to a failure to elicit
a delayed hypersensitivity response, but rather to
be the result of induction of a suppressor T cell
population(s) [50]. The findings of Handman
et al. [52] suggest that the susceptibility of strains
of mice to L. tropica may depend both on a per-
missive macrophage and on defective T cell recog-
nition of parasite antigens on the surface of in-
fected macrophages (for further discussion see
Suppression of Immune Mechanisms below).
Although nothing is known about the genetic
control of resistance to leishmanial infection in
humans, Walton and Valverde [54] have observed
racial differences in the course and outcome of
mucocutaneous leishmaniasis in residents of the
eastern Andes of Bolivia. They found that extreme
facial mutilation due to mucocutaneous leish-
maniasis is almost exclusively confined to persons
of African ancestry, even though many more cases
of leishmaniasis are seen among indigenous
Amerinds in the area. Skin testing with leish-
manial antigen gave greater reactions among
blacks and suggested that mutilation might be due
to necrosis resulting from an exaggerated immune
response.
Pearson et af.
Early Events in Leishmaniasis: The Interaction of
Leishmania Promastigotes with Mammalian Host
Defense Mechanisms
Macrophage-promastigote interactions. Although
Leishmania exist only transiently as promastigotes
in their mammalian hosts, the initial interaction of
the promastigote stage with cellular and humoral
host defense mechanisms is critical to the develop-
ment of infection. In genetically susceptible mam-
mals, the macrophage appears to serve as a
sanctuary in which Leishmania promastigotes
escape the potentially adverse effects of serum and
polymorphonuclear leukocytes and convert to the
amastigote stage.
Several investigators have performed micro-
scopic studies of the interaction of promastigotes
with resident, rodent peritoneal macrophages [55-
59] and tumor cell lines [60] in vitro and have
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shown that promastigotes are rapidly ingested
after macrophages bind them by either their
flagellar or aflagellar poles. Ingestion is followed
by phagosome-lysosome fusion. Promastigote in-
gestion can be inhibited by cytochalasin Band cy-
tochalasin D [58, 59], which interfere with poly-
merization of microfilaments in macrophages and
thereby block the phagocytic mechanism. There is
no evidence that promastigotes by themselves can
actively penetrate the macrophage membrane.
There appear to be "recognition" or "binding"
sites that facilitate the attachment of promasti-
gotes to macrophages. Evidence presented by
Chang [61] indicates that the attachment of L. don-
ovan; promastigotes to rnacrophages occurs
via a receptor-ligand interaction. Experiments in
which promastigotes were treated with glycosi-
dases and competition experiments in which
various monosaccharides were used suggest that
some of the binding ligands are glycoconjugates.
Chang found that promastigote-macrophage
binding was sharply reduced by treating either
parasites or macrophages with trypsin or by incu-
bation with the cation chelator EGTA. Zenian
[62] has reported that attachment of L. tropica
promastigotes to murine macrophages is depen-
dent on various carbohydrates and is inhibited by
EDTA. These studies suggest the existence of a
glycoprotein receptor(s) or binding site(s) on the
parasite andlor macrophage, the activity of which
is dependent on the availability of divalent
cations.
Immunobiology of Leishmaniasis 913

A number of investigators have observed that 50

PM 1011
promastigotes can be killed intracellularly after
ingestion in vitro by rodent peritoneal macro- 40
phages [58, 63-65]. This led to speculation that, in
nature, promastigotes might convert to the more ..,
I
resistant amastigote stage within skin fibroblasts Q 30
and that amastigotes released from fibroblasts x
~
might subsequently infect macrophages. Support e, 20
o
for this hypothetical concept is based on the work
of Chang [66], who demonstrated that a species of
10
Leishmania that produced cutaneous infection in
humans (but not L. donovanii could convert from
the promastigote to an amastigote-like state in
15 30 45 60 75 90 lOS 120 I3S 150 165 180
human skin fibroblasts in vitro. However, Leish-
TIME (MINUTES)
mania have not been identified within fibroblasts
in infected animals or humans, and thus a critical Figure 5. Oxidative response of human monocyte-
piece of data necessary to substantiate this theory derived macrophages exposed to Leishmania donovani.
A luminol-enhanced (1.25 x 1O-6M) chemiluminescence
has not been observed. A much more likely hy-

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assay was used to assess the oxidative burst at a promasti-
pothesis is that rodent peritoneal macrophages gote (PM):phagocyte ratio of 10:1 (. .); amasti-
studied in vitro differ from resident macrophages gote (AM):phagocyte ratio of 10:1 (~); or amas-
in the dermis and that conversion of promasti- tigote.phagocyte ratio of 50:1 (0-----0) in the presence
gotes to amastigotes occurs within macrophages in of 10070 nonimmune, heat-inactivated (at 56 C for 30 min)
serum. Mononuclear phagocytes were studied after five
the skin.
days of cultivation in vitro. Each point represents the cor-
rected mean cpm ± SEM per 1 x 105 macrophages,
based on duplicate determinations. Amastigotes were used
after incubation at 37 C for 4 hr to ensure shedding of
residual hamster membranes as documented by electron
l/l 2000
Q) microscopy. Reprinted with permission from Journal of
>.
o Immunology [70].
o
0'
o
ct 1000
... 800 Pulvertaft and Hoyle [55] were the first to ob-
g 'serve that human mononuclear phagocytes ob-
U
::I
C
oc tained from serous exudates in pathological con-
o ditions or from bone marrow aspirates could
~
ingest L. donovani promastigotes; however, these
o
Q investigators neither quantified their observations
nor assessed the fate of intracellular parasites.
Pearson et al. [67] subsequently demonstrated that
human monocyte-derived macrophages cultivated
for five or more days in vitro bind and ingest
48 72 promastigotes (figure 2), permit their conversion
Hours to an amastigote-like state, and support subse-
Figure 4. Leishmanial infection of human monocyte- quent intracellular parasite multiplication (figure
derived macrophages. Peripheral blood mononuclear 4). Their findings have subsequently been con-
phagocytes were cultivated in vitro for five days and then firmed by Murray and Cartelli [68].
infected with Leishmania donovani promastigotes at a Several investigators have assessed the phago-
parasite.macrophage ratio of 20:1 for 2 hr. Each point cytic oxidative response that accompanies inges-
represents the mean ± SEM of cell-associated parasites
per 100total macrophages, based on four or more experi- tion of promastigotes by macrophages. These
ments done in duplicate. Reprinted with permission from studies followed the observation of Wilson et al.
Journal of Immunology [70]. [69] that Toxoplasma gondii failed to trigger a
914
phagocytic oxidative burst when ingested by hu-
man mononuclear phagocytes, This is not the case
with leishmanial promastigotes, however. Pearson
et al. [70, 71] and Murray and Cartelli [68] have
demonstrated that human mononuclear phago-
cytes can undergo an oxidative burst when ex-
posed to promastigotes (figure 5). Similarly,
Meshnick and Eaton [72] and Murray [73] re-
ported that an oxidative burst accompanies the
ingestion of promastigotes by peritoneal macro-
phages from several strains of mice, whether or
not they are genetically susceptible or resistant to
Leishmania.
The survival of leishmanial promastigotes in
human monocyte-derived macrophages probably
relates in part to the macrophage's relatively low
intrinsic oxidative capacity, lack of myeloperoxi-
dase, or both. Peripheral blood monocytes have a
progressive decrease in oxidative capacity after
several days of in vitro cultivation [74] and lose
lysosomal myeloperoxidase after four days of cul-
tivation [75]. It has been postulated that these fac-
tors may contribute to the survival of T. gondii
and other microbes [76] within mononuclear
phagocytes, and such factors are probably im-
portant in the intracellular survival of Leishmania
as well.
Support for this hypothesis comes from the fol-
lowing observations. Promastigotes survive poor-
ly in monocytes obtained from human blood [68,
71]. This is in contrast to the survival of pro-
mastigotes in human monocyte-derived macro-
phages that are cultivated for five or more days in
vitro before exposure to promastigotes and that
have a diminished oxidative microbicidal capacity.
In addition, Murray and Cartelli [68] demon-
strated that human monocyte-derived macro-
phages, which were activated by mitogen- or anti-
gen (non-leishmanial)-stimulated lymphokines,
produced more H 202 during ingestion of promas-
tigotes than did control macrophages and, in con-
trast to control macrophages, killed ingested pro-
mastigotes. Finally, Murray [77] showed that
promastigotes can survive in a murine macro-
phage-like tumor cell line that is not capable of
producing a phagocytic oxidative response. Lym-
phokine activation of these cells prior to incuba-
tion with promastigotes led to restoration of ox-
idative microbicidal mechanisms and resulted in
killing of ingested promastigotes. These data indi-
cate that the killing of promastigotes by mononu-
Pearson et al.
clear phagocytes correlates with the magnitude of
the phagocytic oxidative burst and suggest that the
oxidative microbicidal capacity of a mononuclear
phagocyte may be an important determinant of its
susceptibility to leishmanial infection.
Conversely, the survival of promastigotes with-
in human monocyte-derived macrophages, which
undergo a phagocytic oxidative burst even though
their response is less potent than that of mono-
cytes or lymphokine-activated macrophages, sug-
gests that promastigotes possess some means of
self-defense against macrophage microbicidal
mechanisms. The Leishmania have an iron-con-
taining superoxidase dismutase that differs from
superoxide dismutases found in mammalian cells
[72]. The leishmanial superoxide dismutase may
represent an adaption for survival in the presence
of activated oxygen products of macrophages in
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vivo. However, the unique properties of the leish-
manial superoxide dismutase may allow for the
development of a specific inhibitor that could be
used for chemotherapy [72].
Although well-endowed with superoxide dis-
mutase [72, 73], promastigotes are relatively de-
ficient in catalase and glutathione peroxidase and
are sensitive to H 20rperoxidase-halide microbici-
dal mechanisms in vitro [72, 73, 78-81]. Murray
[73] showed that promastigotes are killed in condi-
tions in which 10 nM H 202/min is generated from
glucose by glucose oxidase in a phagocyte-free sys-
tem. The addition of a peroxidase with an appro-
priate halide enhanced killing. Pearson and Steig-
bigel [78] also demonstrated killing of promasti-
gotes in the glucose-glucose oxidase system and
found that >50070 of promastigotes were killed
after 2 hr of exposure to 10-5 M H 20 2 •
Furthermore, Gottlieb and Dwyer [82, 83] dem-
onstrated that L. donovani promastigotes are
unique in having acid phosphatase activity distri-
buted over their entire external surface membrane
and within their flagellar reservoir region. The
physiologic role of surface membrane-bound acid
phosphatase has not been established. Its activity
may enable the organism to obtain necessary
nutrients from organic phosphates in the environ-
ment, which would not otherwise be available,
and thereby contribute to the survival of parasites
within the phagolysosomal system of macro-
phages. Surface-bound acid phosphatase might
even in some way protect against macrophage
microbicidal mechanisms.
Immunobiology of Leishmaniasis
Interaction of promastigotes with PMNs.
Pearson and Steigbigel [78] have shown that
PMNs from healthy human donors can ingest and
kill L. donovani promastigotes in vitro. Although
some promastigotes were ingested in the absence
of complement, the presence of complement signi-
ficantly facilitated their uptake. Oxidative micro-
bicidal mechanisms appeared to be responsible for
promastigote killing since (1) the ingestion of
promastigotes was associated with a phagocytic
oxidative burst; (2) promastigotes were shown to
be susceptible to oxidative microbicidal mecha-
nisms; and (3) there was no evidence of parasite
death in PMNs from a donor with chronic granu-
lomatous disease of childhood, a condition in
which leukocytes are unable to produce toxic oxi-
dative metabolites. Further studies are needed to
determine the role that human PMNs play in de-
fense against Leishmania and the manner in which
promastigotes avoid ingestion and killing by these
cells.
It might be conjectured that in naturally ac-
quired infection promastigotes are inoculated at a
cutaneous site where complement either is not
active or is locally inactivated (possibly by factors
in the saliva of the sandfly). Promastigotes would
thus be partially protected from ingestion by
PMNs, and the likelihood that they would safely
reach the confines of a macrophage would be en-
hanced. In contrast to ingestion by PMNs, inges-
tion of promastigotes by mononuclear phagocytes
occurs readily in the absence of immune serum or
complement.
Lethal effects of serum on promastigotes.
Humoral factors also serve as a potential defense
against Leishmania. Several investigators have
found that promastigotes are susceptible to lysis
by both nonimmune and immune serum from
animals [84-86] and humans [87-92]. In 1926
Hindle et al. [87], working in China, demon-
strated that fresh, but not heat-inactivated serum
from healthy donors and from patients with vis-
ceral leishmaniasis destroyed L. donovani pro-
mastigotes in vitro. Subsequently, Adler [88] and
Taub [89] in Israel, Lainson and Strangways-
Dixon [90] in British Honduras, and Ben Rachid
[91] in Algeria reported that sera from donors who
had no history of leishmaniasis killed promasti-
gotes of several Leishmania species. It is possible
that these donors had had prior subclinical infec-
tion and had thus acquired immunity. However,
915
,
INACTIVATED SERUM
~
ts
~
~
~
@ 105
~
l.l.J
~
t3
~
4
10
0 30 60
TIME (minutes)
Downloaded from cid.oxfordjournals.org by guest on April 30, 2011
Figure 6. The effect of fresh and heat-inactivated (at
56 C for 30 min) non immune human serum on promasti-
gote viability. The number of viable promastigotes, as
assessed by flagellar motility, was determined during incu-
bation at 37 C in fresh serum, 10070 heat-inactivated se-
rum, or medium alone. Results are expressed as the mean
effect of 10 sera with brackets enclosing ± SEM. Re-
printed with permission from Journal of Immunology
[92].
sera from North American donors with no history
of exposure to Leishmania are also lethal for
L. donovani promastigotes (figure 6) [92]. Pear-
son and Steibigel have studies the mechanism
responsible for the lethal effect of serum obtained
from these nonimmune donors and have found
that promastigotes in vitro activate the comple-
ment cascade through the classical pathway and
are killed by the complement membrane attack
complex (C5B-C9).
The mode of complement activation, the agglu-
tination of promastigotes exposed to heat-inacti-
vated serum (56 C for 30 min), and the loss of
lethal activity when sera were absorbed with
promastigotes suggested that antibodies were
responsible for activating the complement cas-
cade. Such antibodies against promastigotes may
have developed in these nonimmune donors as the
result of exposure to common antigens shared
with other protozoa, the mycobacteria [93, 94],
other bacteria, or material encountered in the en-
vironment. For example, infection with one mem-
ber of the family Trypanosomatidae can be ac-
companied by production of cross-reacting anti-
916
bodies to other members of the family [19, 95, 96].
This could explain the presence of antileishmanial
antibodies in donors from areas where trypano-
somes and nonmammalian Leishmania are en-
countered, but it is an unlikely explanation for the
presence of antileishmanial antibodies in North
American donors with no known exposure to this
family of protozoa.
Another possible source for these cross-reacting
antibodies against promastigotes might be natural
antibodies that develop in response to antigens
found on mammalian erythrocytes. There are im-
munochemical similarities between leishmanial
antigens and human A-B-O blood group antigens
[97, 98]. There is even some epidemiologic data
suggesting that the distribution of Leishmania
species, as classified by excreted factor serotypes,
may correlate with the frequency of A-B-O blood
groups in populations in endemic regions [97, 99].
The role of antibody, complement, and other
serum factors in defense against leishmania
promastigotes remains uncertain, however. Serum
factors probably provide an important but incom-
plete barrier against infection. Further efforts are
needed to understand the factors that allow para-
sites to reach safely the confines of the mono-
nuclear phagocyte. Some promastigotes may be
taken up by macrophages before they can be killed
by serum, or, alternatively, a subset of promasti-
gotes may be resistant to the lethal effects of com-
plement. Dwyer [100] has shown that promasti-
gotes have the ability to cap antibodies; that is,
antibodies against Leishmania that bind to the
surface of the parasite can be moved into patches
or a single cap on the parasite's surface and, in
some instances, are subsequently shed. However,
capping is a relatively slow process in relation to
complement activation, and it is unlikely that
capping plays a role in protecting promastigotes
from the lethal effect of serum. Alternatively,
saliva or other factors from the sandfly might coat
promastigotes and thereby protect them from
antibody and/or complement, or salivary factors
might even locally inactivate complement. This
possibility has not yet been examined. After the
parasite converts to the amastigote stage, it ap-
pears to be resistant to the lethal effects of nonim-
mune serum [84, 92].
Pearson et al.
Later Events in Leishmaniasis: The Interaction of
Leishmania Amastigotes with Mammalian Host
Defense Mechanisms
At this point we turn our attention to the amasti-
gote stage of Leishmania, which is the stage found
in the mammalian host during infection, and to
the way amastigotes, in contrast to promastigotes,
interact with host defense mechanisms. Once
Leishmania have converted to amastigotes within
macrophages, cell-mediated immune mechanisms
appear to be responsible for controlling subse-
quent infection, although humoral factors may
playa role in modulating cell-mediated responses
during infection and possibly even in protecting
the host from reinfection. Studies of cell-mediated
immune responses during leishmaniasis have in-
volved a number of animal models and multiple
Downloaded from cid.oxfordjournals.org by guest on April 30, 2011
species of Leishmania. Thus, interpretation of the
findings and generalization to humans is difficult
because the immune response can vary as a func-
tion of both the mammalian host and the species
of Leishmania. In general, investigators have
focused on (1) the interaction of amastigotes with
resident peritoneal macrophages from rodents or
with human phagocytes from peripheral blood
studied in vitro (2) the role of cell-mediated im-
mune mechanisms and macrophage "activation"
on amastigote survival; (3) the role of immune
suppression during active leishmaniasis; and (4)
the role of antibody and other humoral factors in
altering the parasite-phagocyte interaction.
Amastigote-phagocyte interactions. Studies of
the interaction of host cells with amastigotes have
focused on tumor cell lines [60, 101-103], poly-
morphonuclear and eosinophilic leukocytes [104],
resident peritoneal macrophages from laboratory
animals that are not natural hosts of Leishmania
[105-113], and human peripheral blood mono-
nuclear phagocytes after various periods of in
vitro cultivation [114]. The effects of macrophage
"activation" on intracellular amastigote survival
will be discussed below.
Canine sarcoma cells have been infected in vitro
with amastigotes or promastigotes of L. mexicana,
L. tropica, and L. donovani [101, 102], and cer-
tain murine macrophage/tumor cell lines have
also been infected with either L. donovani [103] or
Immunobiology of Leishmaniasis
L. mexicana [60]. Once infected, the cell lines can
be used to propagate parasites and to assess the
effects of chemotherapeutic agents on intracellu-
lar amastigotes.
Recent reports by Wyler [115] and Klempner et
al. [116] indicate that attachment of amastigotes
to human mononuclear phagocytes is dependent
at least in part on amastigote factors. Pretreat-
ment of amastigotes with cytochalasins, which are
microfilament inhibitors, decreased the attach-
ment of amastigotes to mononuclear phagocytes
in vitro. However, once amastigotes have attached
to a macrophage, ingestion depends on the macro-
phage's phagocytic apparatus. There is no evi-
dence that amastigotes have the capacity by them-
selves to actively penetrate into macrophages. In
addition, there is evidence from an animal model
that suggests that there may be subpopulations of
macrophages that differ in their ability to bind
and ingest amastigotes and to support subsequent
intracellular parasite growth and replication [111].
Amastigotes are not killed by human monocytes
[68, 71] or by "nonactivated" monocyte-derived
macrophages [114] in vitro. When ingested by
monocyte-derived macrophages, amastigotes elicit
a phagocytic oxidative response, but it is smaller
than that elicited by an equal number of promasti-
gotes (figure 5). Pearson et al. [70] found that the
response to amastigotes was approximately 10070
that to promastigotes as measured in a luminol-
enhanced chemiluminescence assay. Murray and
Cartelli [68] reported similar results using an assay
for release of H 2 0 2 • The reason(s) why the two
parasite stages elicit different responses is not
clear but may relate to differences in motility or
size, to stage-specific differences in exposed anti-
gens, or to differences in attachment of the para-
site to the macrophage. Murray [116a] has also
found that amastigotes contain more catalase and
glutathione peroxidase than promastigotes and are
four times more resistant to enzymatically gener-
ated H 2 0 2 • Once inside macrophages, intracellular
Leishmania neither stimulate continuous oxidative
activity nor do they permanently interfere with
subsequent oxidative responses to other particu-
late stimuli [70].
After amastigotes have gained entrance into ro-
dent peritoneal macrophages [106-108] or human
917
mononuclear phagocytes [114], they multiply in
phagolysosomes. In electron microscopic studies
of rodent peritoneal macrophages infected in
vitro, Alexander and Vickerman [106] demon-
strated fusion of parasitophorous vacuoles con-
taining L. mexicana with secondary lysosomes
labeled with colloid saccharated iron oxide. Chang
and Dwyer [107, 108] confirmed these studies
using hamster peritoneal macrophages and L. dono-
vani amastigotes. They demonstrated fusion of
secondary lysosomes in parasitophorous vacuoles
using thorotrast (an electron-dense marker).
Phagosome-lysosome fusion was subsequently
demonstrated in mice in vivo by Berman et al.
[113]. In this respect Leishmania resemble Myco-
bacterium lepraemurium [117], which permits
phagosome-lysosome fusion in macrophages;
however, Leishmania differ from viable Mycobac-
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terium tuberculosis [118] and T. gondii [119],
whose survival within macrophages has been attri-
buted to their capacity to inhibit the fusion of
parasitophorous vacuoles with lysosomes. Leish-
mania also differ from Trypansoma cruzi, which
escapes from the phagocytic vacuole and resides in
the cytoplasm of the host cell [120].
Leishmania amastigotes must be either resistant
to lysosomal enzymes or somehow protected from
lysosomal contents within the parasitophorous
vacuole. Greenblatt and co-workers [97, 99, 121-
123] have found that amastigotes in vivo and
promastigotes in vitro release a complex poly-
saccharide, termed excreted factor, that can en-
hance the growth of amastigotes in nonpermissive
rodent peritoneal macrophages [121] and of
promastigotes in culture [122]. It is possible that
excreted factor plays a role in protecting intra-
cellular amastigotes from potentially toxic lysoso-
mal constitutents.
The parasitophorous vacuole that contains
Leishmania seems to be part of a phagosome-lyso-
some vacuolar apparatus by which exogenous sub-
stances can be brought into contact with amasti-
gotes via phagocytosis or pinocytosis from the ex-
tracellular menstruum. When Chang and Dwyer
[108] added thorotrast to macrophages previously
infected with amastigotes, the marker was found
within macrophage pinosomes, secondary lyso-
somes, the parasitophorous vacuole, and even in
918
the parasite's flagellar pocket and cytoplasm. The
dynamic phagosome-lysosome vacuolar apparatus
may be the basis for the successful delivery of
pentavalent antimonial compounds to macro-
phages by liposomes [124-126] in experimental
leishmaniasis.
Finally, amastigotes, like promastigotes, are
susceptible to ingestion and killing by human
PMNs and eosinophils [104]. The role of these
phagocytes in preventing amastigotes from gain-
ing access to uninfected mononuclear phagocytes
is unknown. In contrast to promastigotes, amasti-
gotes have not been shown to be susceptible to
killing by serum factors [84, 92].
Cell-mediated immunity and macrophage "acti-
vation." Several animal models and in vitro sys-
tems have been used to investigate the role of cell-
mediated immunity in host defense against Leish-
mania. Many of these studies have been reviewed
in detail elsewhere [19-23, 27, 127]. The impor-
tance of cell-mediated immune mechanisms was
first suggested by studies involving a nonhuman
pathogen, Leishmania enriettii, which produce
spontaneously healing cutaneous ulcers in guinea
pigs [128-133]. Healing of the ulcers was found to
be associated with the development of delayed
hypersensitivity skin reactions as well as in vitro
lymphocyte proliferative responses to leishmanial
antigen. Although specific antibodies against
Leishmania were produced during infection, they
did not result in resolution of the ulcers. Suppres-
sion of cell-mediated immune responses by anti-
serum against lymphocytes increased the severity
of infection [132, 133], an observation suggesting
that the lymphocytes played an important role in
controlling leishmanial infection.
Preston et al. [134, 135] subsequently studied
the role of cell-mediated immunity in an experi-
mental model of cutaneous leishmaniasis in which
CBA mice were infected with a human pathogen,
L. tropica. They demonstrated, using thymecto-
mized, irradiated mice, that the healing of cutane-
ous ulcers required an intact T cell system [134]. In
addition, they found that lymphoid cells obtained
from healed CBA mice or from spleen, peritoneal
exudates, or lymph nodes conferred protection on
recipient syngeneic mice challenged with L. tro-
pica. Serum from healed mice was not protective
by itself, but it did enhance the protective effect of
Pearson et al.
lymphoid cells when administered concurrently
[135].
Further evidence for the role of cell-mediated
immune responses against Leishmania comes
from animal models of visceral leishmaniasis.
Studies by Skov and Twohy [136, 137], in which
immunosuppressive measures were used in C57Bl/
6J mice, suggested that T cells play the predomi-
nant role in acquired immunity to infection with
L. donovani. Rezai et al. [38] later confirmed in
cell transfer experiments that immunity to L. don-
ovani infection was mediated by thymus-depen-
dent lymphocytes. Transfer of immune serum
alone did not confer immunity to recipient ani-
mals. The results from these in vivo animal models
of visceral leishmaniasis were similar to those
from animal models of cutaneous leishmaniasis in
demonstrating the primary role of cell-mediated
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immune mechanisms in controlling leishmanial in-
fection.
The fate of Leishmania within mononuclear
phagocytes appears to depend on the state of mac-
rophage activation. Miller and Twohy [138] ob-
served that L. donovani amastigotes would multi-
ply in vitro in peritoneal macrophages from nor-
mal mice but not from mice previously infected
with the parasite. Their findings suggested that
macrophages became "activated" during infection
to inhibit multiplication of intracellular parasites.
Smrkovski and Larson [139] found that mice
immunized with BCG were protected against a
subsequent challenge with L. donovani amasti-
gotes. Such protection could be abrogated, how-
ever, by treatment with cortisone [140]. In addi-
tion, L. donovan i-infected mice that received
BCG had decreased numbers of amastigotes in
their livers and spleens. The data suggested that
macrophages were nonspecifically activated by
BCG to kill intracellular L. donovani, but, be-
cause species of Leishmania and Mycobacterium
may have antigenic similarities [93, 94], a cross-
reacting, antigen-specific response could not be
excluded. These findings with L. donovani are in
contrast to those of Grimaldi et al. [141], who
found that neither BCG nor the immunostimulant
levamisole resulted in resolution of cutaneous
lesions or in production of a hyperallergic reaction
in C3H mice with cutaneous leishmaniasis caused
by L. mexicana.
Immunobiology of Leishmaniasis
The available in vitro data also also indicate
that the intracellular fate of amastigotes depends
on the capacity of T cells to activate macrophages.
MaueI et al. [142] and Buchmiiller and Mauel
[143] demonstrated that isolated mouse peritoneal
macrophages endocytize L. enriettii amastigotes in
vitro and permit their intracellular survival. How-
ever, cocultivation of infected macrophages with
syngeneic splenic lymphocytes that had been stim-
ulated either by allogeneic cells in mixed lympho-
cyte culture or by the mitogen concanavalin A
(Con A) resulted in intracellular parasite killing.
In addition, exposure to Con A-stimulated lym-
phokines produced a concomitant increase in the
oxidative capacity of macro phages after phagocy-
tosis [79]. Similar results have been reported by
Murray (l16a] and Haidaris and Bonventre [143a]
with L. donovani. These investigators have hy-
pothesized that the destruction of parasites within
activated macrophages might be mediated by en-
hanced oxidative microbicidal mechanisms.
The model of Mauel et al. [142, 143] has been
criticized because L. enriettii does not multiply
within murine macrophages in vitro. This was not
a problem, however, in the studies of Nacy et al.
[144], who defined conditions for the replication
of L. tropica amastigotes in mouse peritoneal
macrophages in vitro and demonstrated that
murine macrophages that were preexposed to anti-
gen-stimulated lymphokines were less likely to
become infected, and that lymphokine-exposed
cells that became infected inhibited replication of
intracellular amastigotes. Macrophages that were
first infected with amastigotes and then treated
with lymphokines exhibited potent microbicidal
activity against intracellular amastigotes. Similar
results were obtained by Handman and Burgess
[145] using murine macrophages that were treated
with granulocyte-macrophage colony-stimulating
factor and exposed to L. tropica and by Haidaris
and Bonventre [146] who studied the interaction
of L donovani with lymphokine-activated murine
macrophages. In the latter study, amastigotes
were killed within macrophages only when the
"activated" state was maintained by daily addition
of lymphokines for several days. Finally, Chang
and Chiao [147] showed that L. donovani infec-
tion of immunocompetent mice elicits the produc-
tion of soluble splenic lymphocyte factors that can
919
"activate" macrophage in vitro to kill intracellular
amastigotes. Thus, in animal models and in vitro
studies of cutaneous and visceral leishmaniasis,
intracellular killing of amastigotes appears to be
dependent on activation of macrophages by lym-
phokines.
The role of cytotoxic mononuclear cells (such as
"natural killer" cells) in protection against infec-
tion with Leishmania is uncertain. In studies by
Bray and Bryceson [128] and Bryceson et al. [129],
mononuclear cells appeared to exert a direct cyto-
toxic effect against guinea pig macrophages that
contained amastigotes of L. enriettii or that were
coated with leishmanial antigen. However, these
findings were not confirmed by others [148].
In some instances, the susceptibility of mice to
leishmanial infection may be due to inadequate
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presentation of leishmanial antigens by macro-
phages to lymphocytes. Handman et al. [52] dem-
onstrated that macrophages from both an L. tropi-
ca-susceptible strain of mice, BALBIc, and a
relatively resistant strain, CBA/H, had leish-
manial antigens on their surface after incubation
with crude antigen preparations, but only BALBI
c macrophages (in contrast to CBA/H macro-
phages) failed to sensitize syngeneic recipient cells
to produce delayed hypersensitivity responses to
the antigen. This failure seemed to be due to re-
duced functional expression of H-2 antigens on
the surface of BALB/c macrophages. As the
authors noted, however, this cannot be the sole
explanation for the variation in susceptibility of
mice to L. tropica, since other L. tropica-resistant
mouse strains (e.g., NZB and C57BI/6), in con-
trast to CBA/H mice, have macrophages that fail
to support intracellular parasite multiplication.
The data raise the possibility that the genetic sus-
ceptibility of mice to L. tropica is associated with
both a permissive macrophage and defective T cell
recognition of parasite antigens on the surface of
infected macrophages [52].
The relevance of these findings to infection in
humans remains conjectural. Berman et al. dem-
onstrated that human monocyte-derived macro-
phages, which support intracellular amastigote
multiplication in vitro [114], have leishmanial
antigens on their surfaces [149]. Murray and Car-
telli [68] recently showed that human mononu-
clear phagocytes could be activated in vitro by
920
mitogen-stimulated lymphokines to kill L. dono-
vani amastigotes. Further studies are needed to
define the critical interaction between human
macrophages (infected with Leishmania) and
lymphocytes in order to determine if there is a
genetic or parasite-induced defect in antigen pre-
sentation to subpopulations of macrophage-acti-
vating T cells in the progressive forms of leish-
maniasis.
Suppression of immune mechanisms. Clinical
experience with human visceral leishmaniasis and
diffuse cutaneous leishmaniasis suggests that cell-
mediated immune mechanisms may be suppressed
during active disease. In both syndromes, delayed
hypersensitivity responses to leishmanial antigen
are absent during infection, but in many patients
they develop following successful antileishmanial
chemotherapy. The best evidence for immune sup-
pression during human leishmaniasis comes from
the work of Peterson et al. [32], who studied four
patients from the Dominican Republic with dif-
fuse cutaneous leishmaniasis caused by a newly
identified strain of Leishmania and found that
adherent cells from peripheral blood could inhibit
lymphocyte-proliferative responses to leishmanial
antigens. None of the patients had delayed cutane-
ous reactions or lymphocyte-proliferative re-
sponses to leishmanial antigens, but they did have
normal proliferative responses to mitogens and to
other antigens, so there seemed to be antigen-
specific suppression in these patients. The total
numbers of lymphocytes and T cells in these pa-
tients were normal. Decreasing the number of
adherent mononuclear cells by passage over nylon
wool or addition of the prostaglandin inhibitor
indomethacin permitted expression of lymphocyte
responses to leishmanial antigens, thereby suggest-
ing the presence of a population of suppressor
macrophages. In contrast to cells from patients
with untreated diffuse cutaneous leishmaniasis,
lymphocytes from patients with localized cutan-
eous leishmaniasis undergo blastogenic responses
to leishmanial antigen during the course of infec-
tion [150].
The findings are similar in human visceralleish-
maniasis, although a suppressor cell population
has not yet been identified. Carvalho et al. [151]
studied the proliferative response of peripheral
blood mononuclear cells to leishmanial antigen
and phytohemagglutinin (PHA) in 14 Brazilian
patients with acute, untreated visceral leishmania-
Pearson et al.
sis and 11 patients who had been successfully
treated one or more years before. In contrast to
the treated patients, infected patients were unre-
sponsive to leishmanial antigen. However, three
of four infected patients became responsive to
leishmanial antigen when retested several weeks
after successful chemotherapy. The lack of a
proliferative response to leishmanial antigen could
not be attributed to reduced numbers of circulat-
ing T cells or to the inhibitory effect of monocytes
or serum factors. Neither infected, treated, nor
control Brazilian patients had depressed responses
to PHA, a fact suggesting that immune suppres-
sion during South American visceral leishmaniasis
is relatively antigen specific.
There is also evidence of broader immune de-
pression during human leishmaniasis. Patients
suffering from Indian visceral leishmaniasis have
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been shown to have depressed lymphocyte respon-
siveness to PHA [152]. Some patients in Ethiopia
with diffuse cutaneous leishmaniasis demon-
strated impaired sensitization to dinitrochloroben-
zene, but they simultaneously had normal delayed
hypersensitivity responses to lepromin and tuber-
culin [153]. Serebryalov et al. [22] have reportedly
found that patients infected with L. tropica before
the third inoculation of diphtheria-pertussis-
tetanus vaccine have a decreased antibody re-
sponse to diphtheria. The significance of these ob-
servations is uncertain because of the variations in
tests and procedures used.
Evidence from animal models further supports
the hypothesis that immune responses in mammals
are suppressed during diffuse cutaneous and
visceral leishmaniasis. L. donovani-infected ham-
sters were reported to have a diminished capability
to reject skin homografts [154] and to produce
antibodies against chicken ovalbumin [155]. Perez
et al. [156] studied the course of cutaneous leish-
maniasis in sensitive (BALB/c) and less suscep-
tible (C57BI/6) mice. They found that infected
BALBI c mice exhibited enhanced responses to
Con A throughout the course of infection, even
though they remained unresponsive to leishmanial
antigen. They also reported that the proliferative
responses to PHA and lipopolysaccharide (LPS)
were depressed. On the other hand, lymphocytes
from C57B1/6 mice had increased DNA prolifera-
tive responses to leishmanial antigen and to the T
cell mitogens PHA and Con A. In subsequent
studies, Perez et al. [157] demonstrated suppres-
Immunobiology of Leishmaniasis
sion of the IgM plaque-forming cell response to
sheep erythrocytes in C57B1/6 mice infected with
L. mexicana at a time during infection when pro-
tective immunity to a challenge infection and de-
layed hypersensitivity responses to parasite anti-
gens were apparently unaffected. During the pe-
riod of maximal immunodepression, spleen cells
from infected mice inhibited the plaque-forming
cell response of spleen cells from uninfected mice.
Scott and Farrell [158] observed depressed proli-
ferative responses to Con A, PHA, and LPS in
BALB/c mice infected with L. tropica. They iden-
tified an adherent cell population that had the
capacity to suppress the in vitro response of
normal spleen cells to Con A. Indomethacin
abrogated the suppression. Although the cell pop-
ulation responsible for suppression was not
specifically identified, the data suggested that
macrophages were responsible.
In contrast, the findings of Howard et al. [48,
50] suggest that the susceptibility of BALBI c mice
to infection with L. tropica is due to induction of
suppressor T lymphocytes that impair a potential-
ly effective, delayed hypersensitivity response. In
their model, delayed skin hypersensitivity was de-
tectable early in infection, but later it was sup-
pressed. When infected mice were subjected to
sublethal irradiation at levels known to deplete the
precursors of suppressor T lymphocytes, infection
resolved at the same rate as in genetically resistant
mice, instead of progressing to the expected fatal
outcome [159]. When irradiated mice were recon-
stituted with normal BALB/c T cells, inexorable
progression of the disease again occurred. Similar-
ly, in studies done by Mitchell et al. [160] in which
hypothymic BALBI c nude mice were reconsti-
tuted with spleen cell suspensions from normal
BALB/c mice, T cell-dependent immune re-
sponses appeared to contribute to the chronicity
of infection and to inhibit healing of lesions.
On the basis of the evidence from mouse models
and from humans it appears that the outcome of
leishmaniasis depends on the complex interplay of
helper and suppressor mononuclear cell popula-
tions, but the exact nature of the suppressor cell
populations remains to be defined.
The role of humoral factors. Markedly ele-
vated immunoglobulin levels and antibodies to
Leishmania are present in human visceral leish-
maniasis, and antibodies are present in cases of
cutaneous leishmaniasis in its various forms [19-
921
27, 161]. In general, the presence and magnitude
of antibody responses in humans are inversely
related to the extent and progression of disease.
As previously discussed, cell-mediated immune
mechanisms appear to be more effective in con-
trolling and limiting the course of infection.
The situation is similar in animal models of
leishmaniasis, in which transfer of immune serum
has not protected nonimmune recipients from in-
fection [38, 39]. In addition, Howard et al. [159]
found that irradiation of L. tropica-infected mice,
at levels that ablated antibody production but not
T-suppressor lymphocytes, had no effect on the
course of infection. Data from Biozzi high- and
low-responder mice likewise suggested that the
presence of antibody against Leishmania did not
result in resolution of infection. Biozzi et al. [162]
used selectivebreeding techniques to develop high-
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and low-responder lines of mice to natural mul-
tideterminant immunogens (e.g., sheep and pi-
geon erythrocytes or flagellar and somatic anti-
gens of salmonellae, bovine serum albumin, and
rabbit gamma globulin). Antibody levels are high
and macrophage activity low in the Biozzi high
responders. The converse is true in low respond-
ers. During L. tropica infection, high responders
develop large, progressive ulcerating lesions and
have high titers of specific antibody. In low re-
sponders the lesions are small and heal sponta-
neously, while antibody titers remain low [163].
The mechanism(s) responsible for the spontaneous
resolution of cutaneous lesions during the immune
phase in this model appears to be independent of
the levels of humoral antibody. As is the situation
in humans, the antibody response correlates in-
versely with the degree and progression of disease.
Even though the presence of antibody against
Leishmania does not result in resolution of viscer-
al, diffuse cutaneous, or mucocutaneous leish-
maniasis in humans or in experimental animals,
serum factors can influence the interaction of
Leishmania with host macrophages and may mod-
ulate cell-mediated immune responses during in-
fection, perhaps by blocking or "masking" impor-
tant antigens. Chang [164] and Herman [165] have
assessed the effects of serum on the interaction of
Leishmania with phagocytes in vitro. Chang found
that rabbit antibodies against amastigotes actually
inhibited the attachment of amastigotes to human
PMNs and monocytes. Herman studied the effects
of serum from C57B1/6J mice that were rein-
922
fected with L. donovani 60 days after an initial in-
oculation of the parasite. He found that serum
obtained 10 to 11 days after reinfection contained
cytophilic antibodies and opsonins for L. dono-
vani amastigotes and promastigotes that resulted
in enhanced binding and ingestion of parasites by
elicited and "activated" mouse peritoneal macro-
phages. Theoretically, enhanced attachment of
amastigotes to macrophages could be either detri-
mental or beneficial to the host, depending on the
intracellular fate of the parasite. The fact that cy-
tophilic and opsonic activity were evident at a time
when spleen and liver parasite burdens were de-
creasing (between 10 and 24 days of infection) sug-
gested that humoral factors facilitated parasite up-
take and subsequent destruction. In contrast, in
the presence of serum obtained from mice 24 days
after reinfection, the percentage of macrophages
able to ingest parasites was decreased to control
levels or significantly below.
Herman's and Chang's data indicate that hu-
moral factors can alter the parasite-phagocyte in-
teraction and that the presence and functional
capacity of antibodies against Leishmania may
vary during the course of infection. Their findings
also demonstrate that serum from animals in-
fected with Leishmania can actually inhibit the at-
tachment of parasites to phagocytes. As pre-
viously noted, Leishmania are able to attach to
macrophages in the absence of antibody and com-
plement. Protein or glycoprotein molecules are
thought to be important in this interaction. Anti-
body against Leishmania may inhibit the attach-
ment of Leishmania to macrophages by masking
important surface molecules and may thereby
shift the mechanism of parasite attachment from
a non-antibody-mediated, spontaneous parasite-
phagocyte interaction to one mediated by Fe re-
ceptors. Alternatively, because Leishmania amas-
tigotes and promastigotes have the capacity to cap
surface antibody [100], they could theoretically
defend themselves by capping or shedding anti-
gen-antibody complexes, which might then block
Fe receptors on phagocytes.
Finally, it seems clear that serum factors may
play an important role in modulating cell-me-
diated immune responses during infection. MaueI
and Behin [37], Preston and Dumonde [135], and
Poulter [39] found that sera from convalescent or
healed animals can augment the level of adoptive
immunity expressed in recipient animals that con-
Pearson et al.
currently receive mononuclear cells from donors
infected with Leishmania. It is possible that circu-
lating immune complexes, lymphokines, or other
serum constituents in addition to antibodies
against Leishmania are responsible for this effect.
Desjeux et al. [166] identified circulating im-
mune complexes and antibodies against IgG, pos-
sibly rheumatoid factors, in patients with severe
mucocutaneous leishmaniasis. There was a close
correlation between the titers of circulating im-
mune complexes and of antibodies against IgG.
Antibodies against IgG were also identified by
Houba and Allison [167] in the sera of African pa-
tients with visceral leishmaniasis. More recently,
circulating immune complexes and antibodies
against IgG (rheumatoid factors) have been identi-
fied in Brazilian patients [168, 169] with South
American visceral leishmaniasis. In a study of 29
Downloaded from cid.oxfordjournals.org by guest on April 30, 2011
patients hospitalized with visceral leishmaniasis in
northeastern Brazil, Pearson et al. [169] found
that 68070 had circulating immune complexes by a
C1q binding assay and 93070 had rheumatoid fac-
tors. The role that rheumatoid factors, circulating
immune complexes, and other serum factors play
in modulating immune responses during human
leishmaniasis deserves further investigation.
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