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Arch Iranian Med 2005; 8 (3): 229 – 233
Case Report
IMMUNOPATHOLOGY OF AUTOLEUKOCYTIC THERAPY OF
CUTANEOUS LEISHMANIASIS
Simin Meymandi MD•*, Shahriar Dabiri MD*, Manzumeh Meymandi MSc*,
Toraj-Reza Mirshekari MD*, Afshin Abdirad MD*, Hossein Nikpour MD*,
Faramarz Soleimani MSc*, Kore Kemp PhD**, Thor G. Theandar PhD**,
Henrick Permin MD***, Arsalan Kharazmi PhD†
This is a report of immunopathological study of our previous work on autobuffy-coat therapy of
dry-type cutaneous leishmaniasis, which focused on cytological activation of macrophages to
eradicate engulfed Leishman bodies by the synergistic effects of lymphocytes, neutrophils, and
inactive monocytes.
We, again, present a case report of skin biopsies which were stained immunohistochemically
before and during autobuffy-coat therapy.
The results revealed increased CD4 and CD8 lymphocytes, CD1a Langerhans cells, interferon
gamma, and TNF-alpha-producing cells during therapy, but IL-10, IL-4, and TGF-beta-positive
immunostained cells were decreased.
We conclude that this kind of therapy induces a Th1-like and suppresses a Th2-like response
in active lesions of dry-type cutaneous leishmaniasis accompanied by overactivity of the
neutrophils and activated histiocytes to eradicate the engulfed Leishmania amastigotes.
Archives of Iranian Medicine, Volume 8, Number 3, 2005: 229 – 233.
Keywords: Autoleukocytic therapy  cutaneous leishmaniasis  cytokines
Introduction
S
urvival and replication of Leishmania in
its safe target macrophages are due to
several evolutionary mechanisms to
modulate host signal pathways of its eradication.1
Different therapeutic regimens—local and systemic—have been used to activate macrophages to
eradicate the engulfed Leishman bodies. Th1-like
response activates this parasitocidal effect and
Th2-like response decreases the intracellular
eradication of the parasite.2
Autologous blood components have been used
in leukemic patients undergoing bone marrow
Authors’ affiliations: *Department of Dermatology, Afzalipour
Medical School, Kerman University of Medical Sciences, Kerman,
Iran, **Department of Immunology, ***Department of Infectious
Disease, †Department of Microbiology, Copenhagen Medical
School, Copenhagen, Denmark.
•Corresponding author and reprints: Simin Meymandi MD,
Department of Dermatology, Afzalipour Medical School, Kerman
University of Medical Sciences, Kerman, Iran.
P. O. Box: 444, Fax: +98-341-2449881
transplantation3 and autologous T cells have been
used as components of photoimmune therapy for
cutaneous lymphoma.4
Continuing our previous work and the first-time
case-reports of autobuffy-coat therapy in dry-type
cutaneous leishmaniasis (DLCL),5 we present
another case report, which focuses on different
immunophenotyping of immune effector cells as
well as cytokines profiles of Th1 and Th2 patterns
during autoleukocytic therapy in skin biopsies.
Case Report
An 18-year-old male suffering from a raised
nonulcerated nodule (measuring 2.5 × 1.5 × 1.5
cm) of the frontal area with a six-month duration
presented to dermatology clinic. Informed consent
of the patient was obtained. Diagnosis of cutaneous
leishmaniasis was based on clinical picture and
characteristic features seen on skin scraping
preparation and histopathological findings on skin
biopsy. A 10-mL sample of peripheral blood was
Archives of Iranian Medicine, Volume 8, Number 3, July 2005 229
Immunopathology of autoleukocytic therapy of cutaneous leishmaniasis
drawn into a heparinized tube, centrifuged at 37˚C
for 15 minutes at 2,500 rpm. Plasma was decanted
and the buffy-coat layer was aspirated into sterile
insulin syringe. A sample of 0.25 – 0.5 mL of
buffy-coat suspension containing 200,000 to
400,000 cells was injected intralesionally by the
dermatologist using multiple injection sites. This
therapy was repeated at one-week interval
injections for three times. At the beginning of the
fourth week, the patient received intralesional
glucantime every other day, for one week. The
lesions were examined clinically for size,
induration, and softness. In addition, punch
biopsies (2 – 4 mm) were taken from the lesion
before therapy (baseline), after two injections (2
weeks), and finally after 4 weeks. Half of the skin
biopsy was fixed in 10% formalin, embedded in
paraffin, and finally stained with hematoxylin and
eosin. The other half was embedded in OCT and
snap-frozen in liquid nitrogen. Frozen sections
were cut and used for immunoperoxidase staining.
Immunoperoxidase staining was CD4 (Dako
M0716, dilution factor = df; 1:10), CD8 (Dako
M0707, df; 1:100), CD1a (Dako M0721, df;
1:100), CD14 (Dako M0852, 1:10), HLA-Dr
(Dako M0704, 1:50), CD19 (Dako M0740, 1:25),
CD56 (Dako M0852, 1:10), INF-gamma
(Genzyme, 1598–00, 1:20), TNF alpha (R&D,
MAB 219, 1:25), IL-12 (R&D, MAB219, 1:25),
IL-4 (Genzyme, 1842–01, 1:10), IL-10 (R&D,
MAB217, 1:20), TGF-beta (R&D, MAB215, 1:20)
and negative control Mouse Ig1 (Dako X 0931,
1:10).
Four individual pathologists scored the
immunostained slides independently. Percentages
of positive immunostained cells among 200 – 400
cell populations in their five most crowded areas
(0.0009 mm2) were counted with high-power fields
(×40) and the percentage of positive cells was
noticed in flow-charts. The data were expressed by
mean ± standard error of mean (sr). Th1 response
was calculated by the mean of the summation of
positive cells of INF-gamma, TNF-alpha, and
IL-12; and Th2 response as the mean of summation
of positive cells of IL-4, IL-10, and TGF-beta. The
lesion showed a beneficial subjective and objective
clinical responsive to the therapy.
Skin scraping slides showed infiltration of
lymphocytes, inactive macrophages, and neutrophils between the anergic collections of the
histiocytes which engulfed by Leishmania
amastigotes (Figure 1). The number of Leishmania
amastigotes was dramatically decreased with some
230 Archives of Iranian Medicine, Volume 8, Number 3, July 2005
Figure 1. Disruption of degenerated macrophage
(arrow-head) and releasing of their engulfed
Lishman bodies (short arrow). Peripheral rimming
are neutrophils, lymphocytes, and macrophages
(×1000, Giemsa stain).
degenerative changes of activated macrophages
(Figure 2).
By looking at the graphs of the immunophenotyping pattern of positive immunostained cells
during therapy and comparing them with baseline
data, we concluded that CD4+ and CD8+
lymphocytes were increased to 3 – 4 times more
than before therapy (Figure 3). CD1a was 1.5 times
increased. INF-gamma and TNF-alpha were
increased to 2 times the level prior to therapy. IL12 was increased as well. IL-4, IL-10, and TGFbeta were decreased to one-half. CD19, and CD56
were negative both before and after therapy.
Discussion
Different local6, 7 and systemic8, 9 therapeutic
regimens have been used to treat cutaneous
leishmaniasis. On the other hand, mechanisms of
parasitic control and evasion of immune response
Figure 2. Penetration of lymphocyte within
ruptured parasitophorous vacuoles. Nuclear and
cytoplasmic degeneration of macrophages are
noticed (×1000, Giemsa stain).
S. Meymandi, S. Dabiri, M. Meymandi, et al
CD4
CD8
INF-gamma
Positive immunostained cells
100
CD1a
IL10
80
IL4
TGF-beta
60
TNF-alpha
IL12
40
20
0
Before
2 w eeks
4 w eeks
Figure 3. Graphs of the immunophenotyping pattern of positive immunostained cells.
are so complicated that the parasite knows how to
retreat into “safe target cells”, actively suppress the
synthesis of reactive oxygen or nitrogen
intermediates, modulate the host cytokine
response, inhibit antigen presentation, and T cell
stimulation.10 Immunotherapy has been shown
successful in the eradication of the disease.
Sjolander et al showed that, in murine
leishmaniasis, vaccination with plasmid DNA
encoding the host protective L. major parasite
surface Ag-2 primed for an essentially exclusive
Th1 response protected mice against L. major
infection.11 Handman et al mentioned that
prophylactic DNA vaccination shifted the T cellderived cytokine environment with an increase in
the INF-gamma-producing Th1 type cells.12
Cabrera et al showed that by using live
Mycobacterium bovis BCG and heated killed
L. amazonensis, one could induce Th1 response
and clinical cure mediated by INF-gamma.13
According to our findings, autoleukocytic
(buffy-coat) therapy increases the percentage of
CD4+ and CD8+ lymphocytes and, from the point
of view of their cytokine profile patterns, induce a
more prominent Th1-like response than Th2-like
response. Gaafar et al demonstrated a Th1 response
in mild form and a Th2 response in nonhealed
severe disease.14 Ajdary et al showed Th1
responses in active and recovered lesions and Th2
response in non-healed lesions.15 Furthermore, the
presence of circulating effector immune cells in the
peripheral blood of cured cutaneous leishmaniasis
patients has been demonstrated.16, 17 In our series,
CD4+ and CD8+ lymphocytes, Langerhans cells,
INF-gamma, and TNF-alpha are increased, while
Leishmania amastigotes, macrophages, IL-4 , IL10, and transforming growth factor beta are
decreased.
Salaiza-Suazo et al demonstrated that patients
with diffuse cutaneous leishmaniasis caused by
L. mexicana have Th1 conditions if treated by two
leishmanicidal drugs (pentamidine and allopurinol)
in combination with recombinant INF-gamma.
Additionally, parasites decreased dramatically,
macrophages diminished concomitantly, while IL12-producing Langerhans cells and INF-gammaproducing NK and CD8+ lymphocytes increased.18
Kolde et al revealed that interferon therapy in
murine leishmaniasis could induce a Th1 response
which activates macrophages to kill intracellular
parasites and leads to a decline of less mature
macrophages in the infiltrate.19
In our series, the increased number of
Langerhans cells and IL-12 cytokines concur with
the findings of others. Li et al also documented
that the treatment of nonhealing lesions in murine
leishmaniasis through combination therapy of
antimonial drugs with INF-gamma depends on the
mechanisms of IL-12 and nitric oxide production.20
Kole et al showed that reducing parasite numbers,
reduced IL-4 but increased IL-12 and inducible
nitric oxide synthase are due to the synergistic
effect of INF-gamma and mannosylated liposomal
doxorubicin in the therapy of experimental visceral
Archives of Iranian Medicine, Volume 8, Number 3, July 2005 231
Immunopathology of autoleukocytic therapy of cutaneous leishmaniasis
leishmaniasis.21 Kropf et al showed that in murine
leishmaniasis, although IL-4 polarized Th2
response, the induction of healing displayed a
strongly elevated response to IL-12.22
TGF-beta is a multipotential cytokine with
diverse effects on immune cells, including the
down-regulation of certain macrophages and the
blockade of INF-gamma-induced macrophage
activation.23 Li et al showed that using anti-TGFbeta treatment promotes rapid healing of murine
leishmaniasis through enhancing in vivo nitric
oxide production by activated macrophages.24
Finally, our data show that autoleukocytic
therapy is an effective and safe mode of therapy
for active cutaneous leishmaniasis. Further
investigations are mandatory to find out the
possible effects of recruitment cytokines on
immune effector cells.
8
9
10
11
12
13
Acknowledgment
We thank Mrs. Lis Christensen for excellent
immunostaining of frozen specimen and Mrs. Azeri
and Mrs. Gharaei for typing the manuscript. This
research is funded through a grant from DANIDA
and the support of the Clinical Microbiology
Department, Copenhagen Medical School,
Denmark.
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