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Parasitología al día

versión impresa ISSN 0716-0720

Parasitol. día v.23 n.1-2 Santiago ene. 1999

http://dx.doi.org/10.4067/S0716-07201999000100001 

TRABAJO DE INVESTIGACION

FREQUENCY EPIDERMAL LANGERHANS CELLS IN SITE
OF INTRADERMAL INJECTION PROMASTIGOTES OF
Leishmania braziliensis IN MICE

Ana Lugo de Yarbuh*

 

*Investigaciones Parasitológicas Dr. "J. F. Torrealba". Departamento de Biología, Facultad de Ciencias, Universidad de los Andes, Mérida 5101 Venezuela. Corresponding: Edificio A, La Hechicera, Dpto de Biología, Facultad de Ciencias, Universidad de los Andes, Mérida 5101 Venezuela.
Telf –Fax (58-74) 401285.
E-mail: lana@ciens.ula.ve

ABSTRACT

We utilized immunostaining assay with Avidin Biotin Peroxidase (ABP) technique and primary rat monoclonal antibody NLDC-145 specific for mouse dendritic cell, characterized Langerhans cells (LC) in epidermis sheet of 1 mm2 of skin of the footpads of inbred females BALB/c mice, intradermally (id) injected with 1.103 cultured promastigotes of Leishmania braziliensis. The result showed that the injection of the parasites in the skin of the animals produced a progressive increment of epidermal LC in the site of the previous injection, and statistically significant (P<0.05) in each study period. The density of epidermal LC was going up by parasite insult in early time, after the 15 minutes (m) post-infection (pi) with 162±31.2 LC/mm2, reaching a maximal value of 4503±713 LC/mm2 to 2 week (w) pi until 898± 481 LC/mm2 to 6 w pi. The number of LC was always higher in epidermis sheet of mice infected with L. braziliensis, than in the epidermis of the control healthy mice, these skin samples showed 120±28.9 LC/mm2. Morphological changes of the promastigotes injected could to be detected in skin Giemsa-stained imprints, the parasite showed form ovoid and a short flagel, between 15 m and 2 hour (hr) pi. No parasites were even seen in the imprint samples than of any other time of infection. These samples also showed many inflammatory cells, such as: activated macrophages (33%) to 15 m pi, neutrophils (46.33%) to 4 hr pi, eosinophils (2.33%) to 4 hr pi, lymphocytes (15.67%) to 6 hr pi, degraded lymphocytes (8.33%) to 1 w pi, monocytes (4.33%) to 5 day (d) pi and activated monocytes (19.33%) to 1 d pi. The stained sections of skin inoculated, revealed amastigotes into macrophages dermal near of the perivascular area and inflammatory process in the dermis consisted of lymphocytes, monocytes, plasma cell and polymorphonuclear cells between 1 w and 6 w pi. No parasites were detected in the epidermis. The results showed that the promastigotes in the skin survived the first 2 hr out of the macrophages, and on the other hand, stimuled various cell types in site of injection of the parasites, and the proliferation of antigen presentation by epidermal Langerhans cells, necessary for the initiation of the specific T cell immune response.
Key words: Leishmaniosis, Epidermis, Langerhans cells, Leishmania braziliensis

INTRODUCTION

American cutaneous leishmaniasis (ACL) is caused by protozoan parasites of the genus Leishmania, and is transmitted by Phlebotominae sandflies, to penetrate the stylets of the biting fascicle until 130 mm for blood sucking, depositing the parasite into epidermis or superior cover of the dermis of mamalians host when it is inoculated natural o experimentaly. In consequence, the contact between the parasites and cutaneous cells, causes a wide spectrum of disease and immunoregulators process, with the participation of LC and other presentation antigen cells-specific T cells. 1,3. The contact of the parasites with the cutaneous cells induce activation of epidermal immunocompetents cells, and different immunoregulators processes in animals susceptible or resistent to the infection, the promastigotes enter into mononuclear phagocytes and are transformed into amastigotes, producing and a wide spectrum of disease in mammalian host.4,5 Some autors have showed that in ACL a significant numbers of LC are enconunter in the infected skin and in the dermis, they have the capacity to take up parasites, the first cells the parasite encounters are those of the skin that internalize the parasite to the draining lymph node, inducing an antigen-specific cell T, and a early immune response that might be relevant to the outcome of the disease.6,7 Factors such as local differences in skin temperature, lymphatic microvasculature or the distribution of epidermal Langerhans cells were hypothesized to contribute to site-specific differences in the severity of a cutaneous Leishmania infections.8,9 Some work has shown that the numbers of epidermal LC are often increased in cutaneous lesions in the model murine,10 in the same model animal, other investigations show a significant decrease in the number of LC in the epidermis at later stages of chronic inflammation, overlying the parasite containing dermal infiltrate and a concomittant increase of LC in this area of the dermis, some of which containing parasites of L. major, this suggests that LC migrates from the epidermis into the dermis prior to the uptake of parasites.11

In the present study, we examined the frequency of epidermal Langerhans cells in epidermis sheet of the rear limbs BALB/c mice, in the site of the injection in early time of infection with cultured promastigotes of Leishmania braziliensis, using ABP techniques. In addition we evaluated the activation of phagocytic cells in the skin of BALB/c mice infected, the morphological changes of the cultured promastigotes when they are deposited into of the skin of the footpads of the mice inoculated and Leishmania antigen in the infected.skin.

MATERIALS AND METHODS

Animals and infections
Sixty female BALB/c mice 6 weeks old, were separated into twenty groups. Three animals from each group were id injected in the left footpads with 1x103 cultured promastigotes of L. braziliensis H/HOM/Ve/93-MAMU93 strain.

Biopsies and samples separation
The infected animals were anesthetized with cloroformo and three skin biopsy samples were taken of the footpads inoculated, to 15 m; 30 m; 1, 2, 3, 4, 5 and 6 hr; 1, 2, 3, 4, 5 and 6 d; 1, 2, 3, 4, 5 and 6 w after inoculation. One sample was used for Giemsa-stained imprints to shows the cellular patterns in the site of infection. Another was fixed in 2% formaline and embedded in paraffin, sections of 7 µm were stained by Giemsa-Colophonium for histopathological12 and identification of Leishmania antigen for immunofluorescense antibody test (IFAT)13 and immunoperoxidase indirect (PAP).14 The other sample of skin was cut into 1 mm2 pieces, and the epidermis used by ABP technique

Avidin Biotin Peroxidase technique
For epidermis separation, 1mm2 pieces of skin were immersed in buffered EDTA 0.02M, in PBS pH 7.2, for 120 m at 37ºC. The epidermis was removed from the dermis using wooden toothpicks, and fixed with acetone for 5 m, the epidermal pieces were transferred to round-botton microplates, hydrated in PBS and sequentially incubated with a primary rat monoclonal antibody NLDC-145 diluited 1:25 for 1 hr15, and secondary antibody biotinylated goat anti-mouse (Amersham) 1:30 for 45 m. A positive reaction was shown by means of incubation with ABP complex (Vector Laboratories Inc.) for 30 m, followed for 3-amino-9-Ethyl-Carbazole (AEC) in 5 mL N, N, Dimethyl-Formamide (Sigma), (500 m L of AEC in 50 m L H202 3% and Acetate buffer 0.1 M to pH, 5.2) for 10 m. Five minute washes with PBS were done between incubations. The epidermal sheets were then mounted on glass slides with glycerin-gelatin solution. The primary antibody was replaced by normal mouse serum or by PBS solution in the epidermis of mice infected and healthy used as control negative.

Cell quantification
The LC were counted in twenty fields from each epidermal sheet at a magnification of 400 X, and the numbers of LC were expressed with the mean LC/mm2 ± standard desviation (S.D.).

Statistical analysis
The data analysed using Scheffe16 technique or analysis of variance to compare the frequency of LC at all time infection.

RESULTS

Few minutes after id inoculation of promastigotes of L. braziliensis into footpads of BALB/c mice, it produced an increase of epidermal LC in the site of injection of the parasites statistically significant (P<0.05) in each study period. The quantification of LC in each epidermal sheet showed an initial value of 162 ± 31.2 LC/mm2 to 15 m, 200 ± 83.7 LC/mm2 to 30 m, 280 ± 70 LC/mm2 to 1 h, 442 ± 22.7 LC/mm2 to 2 h, 443 ± 35.9 LC/mm2 to 3 h, 453 ± LC/mm2 tto 4 h, 525 ± 43.8 LC/mm2 to 5 h, 543 ± 102 Lc/mm2 to 6 h, 899 ± 203.8 LC/mm2 to 1 d, 1448 ± 128 LC/mm2 to 2 d, 2558 ± 357 LC/mm2 to 3 d, 2622 ± 170 LC/mm2 to 4 d, 3062 ± 98.1 LC/mm2 to 5 d, 3285 ± 120 LC/mm2 to 6 d, 3858 ± 225 LC/mm2 to 1 w and, 4503 ± 713 LC/mm2 to 2 w. These values start to decrease after the 2 w pi, showing the epidermis 3787 ± 225 LC/mm2 to 3 w, 1865 ± 356 LC/mm2 to 4 w, 1454 ± 162 LC/mm2 to 5 w, until falling to 898 ± 481 LC/mm2 to the 6 w pi. However, the values of epidermal LC remained higher during the study period, and always highest in experimentally infected mice than in twenty healthy mice, in these animal controls the number of epidermal LC varing between 95 ± 27.2 LC/mm2 to 121 ± 29.5 LC/mm2. Figure 1 show the frequency of the epidermal LC detected in each group of three mice infected for each time infection and the number of epidermal LC in twenthy healthy mice. Figure 2 show LC in epidermis sheet of footpads of mice with 2 w pi.

Figure 1. Epidermal Langerhans cells in footpads of BALB/c mice infected with Leishmania
braziliensis
promastigotes

Figure 2. Langerhans cells in epidermis of BALB/c Leishmania braziliensis
infected (2 week after infection). Avidin biotin immunoperoxidase using monoclonal
antibody NLDC-145 (magnification 250 X).

The Giemsa stained smears of skin inoculated showed 13.33% normal macrophages (NM), 78.33% activated macrophages (AM), 5.33% neutrophils (N) and 3% lymphocytes (L) to 15 m pi. The NM, AM and N were the cells more abundant in the infected skin in all time infection. The eosinophils (E) (2.33%) were detected only to 4 h pi. The highest percentages of lymphocytes (L) (15.67%), degraded lymphocytes (DL) (8.33%), monocytes (M) (4.33%) and activated monocytes (AM) (19.33%) were detected to 6 h, 1 w, 5 d, and 4 h pi respectively (Table 1).

The histological sections showed small clusters of parasitized macrophages, mononuclear and polymorphonuclear cells.

In immunoperoxidase-stained sections parasites were stained a brown. The Leishmania antigen was identified in the cytoplasm and cell membrane of macrophages, in the epidermis and in the follicular dendritic cells. The IFAT showed intense fluorescence in the dermis near the dermis folicular and in peripheral vascular area.

Table 1. Cell population in skin of samples of the footpad of BALB/c mice infected with Leishmania braziliensis promastigotes.

Time
NM
%
AM
%
N
%
E
%
L
%
DL
%
M
%
AM
%

15 m
13,33
78,33
  5,33
0
3
0
0
0
30 m
28
74,3
  5
0
  1,3
0
0
0
  1 h
25,67
68
  1,67
0
1
0
     0,33
0
  2 h
29,66
57,66
12,67
0
0
0
0
0
  3 h
31
40,67
10,67
0
    3,66
0
     0,33
0
  4 h
10,33
19,67
46,33
    2,33
0
0
0
       19,33
  5 h
30,66
15,95
33,12
0
0
0
0
     31,6
  6 h
46,66
15,33
31
0
       15,67
0
0
       15,65
  1 d
50
38,33
10,66
0
0
0
0
0
  2 d
51,33
33
  7
0
0
3
0
0
  3 d
63
32,33
  0,3
0
0
0
0
2
  4 d
71,33
28,66
  0,3
0
0
0
0
0
  5 d
64,33
23,67
  0
0
0
0
     4,33
0
  6 d
70
27
  2
0
3
0
0
0
  1 w
62
22,67
  3,33
0
0
     8,33
0
     3,67
  2 w
76,67
14,33
  0
0
3
0
0
0
  3 w
83,51
16,39
  0
0
    2,99
0
0
0
  4 w
63,76
34,61
  0
0
0
0
0
0
  5 w
56,53
42,74
  0
0
    2,17
0
0
0
  6 w
68,25
31,74
  0
0
0
0
0
0
HM
96
  0
  2
1
1
0
0
0
HM
97
  0   0
0
2
0
1
0

%: Percentaje of the value espressed as mean of cells/mm2
NM: Normal Macrophagas; AM: Activates Macrophagas; N: Neutrophils; E: Eosinophils; L: Lymphocytes; DL: Degraded Lymphocytes;
M: Monocytes; AM: Activated Monocytes
HM: Helthy mice; m: minutes; h: hour; d: day; w: week

DISCUSSION

The clinical manifestations of ACL can be reproduced experimentally in different strains of mice. In healthy mice it has shows a positive correlation between LC and dendritic epidermal T cells (DETC) numbers, however this correlation was not observed in L mexicana infected mice, suggesting that the infection alters the balance between the two cells types and the agents that modulate LC and DETC cell densities change the development of experimental leishmaniasis.6 In cutaneous lesion, it had been showing factors such as the strain of parasite and model animal used, as well as, the site of infection, can remarkably influence lesion development and differences in the epidermal LC density detected an increase of the epidermal LC in the susceptible mouse models,10 while17 observed a pronounced decrease of the number of LC in the epidermis overlying the parasite containing dermal infiltrate and a concomittant increase of LC in this area of the dermis, some of which contained parasite in mice infected with L. major. In this study, we showed that only an application of promastigotes injection of L. braziliensis in mice, produced continuously progressive changes in the skin cells, determinated by the numbers of LC that migrate to tissue infected. These variations in the LC and other epidermal immunocompetent cells in the immune response to parasites in cutanous lesions of human host and infected animal, suggest the critical role these cells hold in the processes involved with infectious diseases. We observed an early epidermal compromise in the leishmanial infection of mice BALB/c. The number of epidermal LC were always higher in skin of the infected mice since 15 m the infection in relation with the LC detected in epidermis sheet the footpads of BALB/c mice healthy. Although differences in host genetics have been shown to be important factors in determining the severity and outcome of an Leishmania infection, and has been documented that factors such as the strain of parasite used, and site of infections, can remarkable influence lesion development.18-19 On the other hand,5 found that cutaneous Leishmania infection can lead to a wide degree of variability in the severity of disease, depending factors suchs as local differences in skin temperature, lymphatic microvasculature, or in the distribution of epidermal LC. We noted that LC production at the footpads of BALB/c infected mice with promastigotes, coincided with the increase of AM, L, M and polymorphonuclear cells in the skin of mice until four weeks pi.

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Acknowledgements. We thanks Dr. F. Tapia Instituto de Biomedicina, Universidad Central de Venezuela, Caracas, Venezuela, for p[roviding the monoclonal antibody NLDC-145.

Financial support. Was given by CDCHT-ULA. Grant C-642-94-03-A and C-747-95-03-B.