Mast cells at the host–pathogen interface: host-protection versus immune evasion in leishmaniasis

Abstract

Infection of a susceptible host with Leishmania, a protozoan parasite, causes the disease leishmaniasis, which is characterized by neutrophil, eosinophil, macrophage, lymphocyte and mast cell infiltration into the infected tissue followed by parasite growth. Although the roles played by other cells in leishmaniasis are known, the role of mast cells remains to be ascertained. Here, we demonstrate that Leishmania regulates mast cell infiltration to the site of infection, mast cell production and mast cell function resulting in differential growth of the parasite in resistant (C57BL/6 or CBA/T6T6) and susceptible (BALB/c) macrophages. An interleukin-3-dependent augmentation in mast cell committed progenitors is observed in BALB/c but not in C57BL/6 mice during Leishmania infection. The mast cell supernatants inhibit IFN-γ-dependent restriction of Leishmania growth in macrophages in BALB/c mice whereas the reverse phenomenon occurs in C57BL/6 mice. Our data reveals a different facet of host–pathogen interaction.

INTRODUCTION

Leishmania, a protozoan parasite that lives primarily within macrophages, causes the disease leishmaniasis. Leishmania donovani, the species that causes visceral leishmaniasis (VL), disseminates to spleen, liver and bone marrow (BM), whereas L. major, the species that causes cutaneous leishmaniasis (CL), remains in the cutaneous lesion and the draining lymph node [1]. In experimental infections, both the resistant and susceptible mouse strains react by recruiting neutrophils, eosinophils, macrophages, lymphocytes and mast cells [2–5] to the site of infection as soon as the promastigotes are inoculated. Of all these cell types, the differential roles played by mast cells in Leishmania infection in a susceptible and a resistant mouse strain remain unknown. A previous study on genetically mast cell-deficient mice with mutations in c-kit (c-kit) or steel (c-kit ligand) loci [6] demonstrated that these mice developed a smaller lesion and healed quicker than their wild-type littermates suggesting a role of mast cells in the promotion of infection [7]. Since the mutated mice were semisyngeneic with C57BL/6 mice, displaying a self-curing phenotype, the mast cells in the resistant mouse were either antiparasitic or their pro-parasitic roles were masked by other factors that dictated resistance [8]. Thus, the previous study failed to suggest any conclusive roles of mast cells in the control of parasite infection [7,9]. Furthermore, since L. donovani disseminates to lymphoid organs as opposed to L. major infection, which is restricted to skin lesion and the draining lymph nodes, mast cells may play different roles in VL and CL. Therefore, a comparative study between susceptible (BALB/c) and resistant strains (C57BL/6 and CBA/T6T6) of mouse was carried out to elucidate the role of mast cells at the host–pathogen interface.

MATERIALS AND METHODS

Leishmania parasite and infection in mice

Leishmania donovani (Strain AG83) was maintained in RPMI-1640 supplemented with 10% FCS [10]. Virulence of the parasite was maintained by serial passage through BALB/c mice [10]. Mice infected with L. major (MRHO/SU/59/NEAL_P (LV39); 2 × 10⁶/mouse) were examined every week for lesion development. The lesions that developed were measured with a direct reading caliper gauge (GMH-390-T, Gallenkamp, London, UK) in two perpendicular diameters. The average diameter (mm) was recorded.

Histopathology of spleen and skin

Tissues were processed for histopathology using light microscopy and electron microscopy following standard protocols. Briefly, the tissues were fixed in Carnoy's fixative, embedded in paraffin, stained with haematoxylene-eosin or with alcian blue-safranin, mounted and observed under a light microscope following standard protocols. For electron microscopy, glutaraldehyde/osmium tetroxide-fixed and resin-embedded tissues were sectioned by an LKB-III ultramicrotome, stained with uranyl acetate and examined in a Jeol-100-CX or Jeol-1200-EX transmission electron microscope.

T cell isolation

T cells were isolated by passing the splenocytes or lymph node cells through nylon wool columns as described earlier [10]. The T cells were then stimulated with ConA (4 µg/ml) for 72 h, supernatants collected and the any residual ConA was neutralized by α-methyl-mannopyranoside and was stored at −70°C till use.

Mastocytosis by sera and T cell supernatants in vitro

The femoral and splenic cells from Leishmania-infected and uninfected mice were cultured at 5 × 10⁴ cells/ml on methylcellulose in presence of sera drawn from different mice, as indicated, or with T cell supernatants from different days of infection in complete medium for 21 days at 37°C, 5%CO₂. The cells were then fixed and stained with toluidine blue as described [11,12]. In some experiments, the T cell supernatants were neutralized by the addition of anti-IL-3 neutralizing antibody (10 µg/ml). IL-3 neutralized T cell supernatants were then used for mast cell inducing activity.

Mast cell isolation, degranulation and parasite load modulation in macrophages by mast cell supernatants

Mast cells were purified from the spleens of infected BALB/c mice by modification of the method described by Wells and Mann [13]. Briefly, the splenocytes, depleted of macrophages, B cells and T cells, were subjected to a percoll-gradient at 2000 r.p.m. for 15 min at 4°C. The cells in the pellet were recovered, washed and suspended in RPMI-1640 complete medium. 0·5 ml of mast cell suspension at a concentration of 10⁷ cells/ml was cultured with 0·5 ml of serum drawn from 120 days postinfection BALB/c mice in presence of crude soluble leishmanial antigen (CSA) at 25 µg/ml. The mast cell supernatants thus generated were tested for their leishmanicidal activity in macrophages as described previously [14]. Briefly, 10⁴ macrophages were cultured with mast cell supernatants, as indicated, for 24 hours, followed by infection with 10⁵L. donovani promastigote for six hours and culture for 72 hours as described earlier. The cells were then fixed, Giemsa-stained and counted under a light microscope. In some cases, mast cells were also counted by staining with Giemsa-colophonium stain, by alcian blue-Safranin stain and also by using the antibodies against mast cell protease-I and protease-II. The profile of mast cell changes remained similar irrespective of the staining method.

Statistical analysis

For in vivo experiments, a minimum of five mice per treatment group was used. Triplicate cultures per treatment were set in vitro. The experiments were repeated at least three times. The significance of difference between the mean values was determined by Student's t-test.

RESULTS AND DISCUSSION

Mast cell number increases during Leishmania infection

The mast cell count per 100 nucleated cells increased significantly (P < 0·001) in BALB/c but not in C57BL/6 mice after L. donovani infection (Fig. 1a). Similarly, mast cell numbers increased significantly (P < 0·001) in the upper dermis in BALB/c (Fig. 1b,c) but not in CBA/T6T6 mice (Fig. 1d) in L. major infection. However, the number of degranulating mast cells was higher in CBA/T6T6 during early L. major infection (Fig. 1e,f) suggesting that the resistant and susceptible mouse strains might have a different mode of mounting the anti-leishmanial immune response through differential regulation of mast cell origin and function.

Fig. 1.

Mast cell number increases during Leishmania major and Leishmania donovani infection in susceptible but not in resistant mouse. (a). BALB/c (○) and C57BL/6 (∇) mice were infected with L. donovani promastigotes (2 × 10⁷/mouse; i.v). Mast cell number in spleen was assessed by toluidine blue staining at the indicated time points after infection. The data presented is mean ± SD (*P < 0·001) and is a representative of one of three individual experiments (n = 5). (b). BALB/c mice, uninfected (Nv) and L. major-infected (Inf), were sacrificed five weeks after infection and processed for detecting mast cells (arrows). The tissues were fixed in Carnoy's fixative, embedded in paraffin, stained with haematoxylene–eosin or with alcian blue-safranin, mounted and observed under a light microscope following standard protocols. (c, d). BALB/c (c) and CBA/T6T6 (d) mice were infected s.c. with L. major. Alcian blue-safranin staining at the indicated time points after infection assessed mast cell number in the upper (○) and lower (∇) dermis. The data presented is mean ± SD (*P < 0·001). (e). Number of degranulating mast cells in the upper dermis of BALB/c (∇) and CBA/T6T6 (○) mice was assessed. The data presented is mean ± SD (*P < 0·001). (f). Intact and degranulating mast cells in the upper dermis of CBA/T6T6 mouse are shown by electron microscopy. (Inf = Infected; M = Mast cells (shown by arrows); Nv = Naïve.)

Differential regulation of mast cell committed progenitors (MCCP) during L. donovani infection

As L. donovani infection disseminates to bone marrow, we tested the mast cell committed progenitor activity in VL. Since the sera from L. donovani-infected BALB/c mice induced myelopoiesis in syngeneic recipients [10], we tested whether or not these sera induced mast cells in vitro. Normal bone marrow cells were induced with sera drawn from mice after different days of infection. It was observed that the sera from 120 days infected mice induced the highest number of mast cells (Fig. 2a). In order to check the number of mast cell committed progenitors (MCCP) in bone marrow and spleen, the cells from these two organs from different stages of infection were cultured in vitro with the sera from 120 days infected mice. It was observed that the maximum number of mast cells was obtained with 120 days infected bone marrow (Fig. 2a). Therefore, together these data suggested that the peak mast cell inducing activity that increased the mast cell committed progenitors in bone marrow and spleen occurred around 120 days after L. donovani infection in BALB/c mouse.

Fig. 2.

Mast cell production in bone marrow and spleen is augmented during L. donovani infection. (a). L. donovani-infected mice sera-induced MCCP differentiation to mast cells in vitro. Sera were collected from BALB/c mice at the indicated time points (X-axis) after L. donovani infection. Bone marrow cells, depleted of macrophages, B cells and T cells, from both naïve and 120-days infected BALB/c mice were cultured with sera for 21 days. Medium was replenished every five days. The numbers of mast cells generated in vitro were estimated. The result, shown as mean ± SD (*P < 0·001), is from a representative experiment that was performed three times. (b) T cell-induced MCCP differentiation to mast cells during L. donovani infection. The splenic (SPL) and lymph node (LN) T cells were purified from BALB/c mice on the indicated days after infection and the supernatants were generated as described earlier. Naïve and 120-days-infected bone marrow cells, depleted of macrophages, B cells and T cells, were cultured for 21 days with the T cell supernatants to generate the mast cells in vitro as described earlier. Media including the T cell supernatant were replenished every fifth day. The mast cells were counted as described in materials and methods. The result, shown as mean ± SD (*P < 0·001), is from a representative experiment that was performed three times. (Inf., Infected; Nv, Naïve). (c) The major mast cell inducing factor in the lymph node T cell supernatant is IL-3 as observed in the mast cell induction assay in vitro by neutralizing the supernatants with a neutralizing anti-IL-3 antibody. The result, shown as mean ± SD, is a representative of three experiments. (*P < 0·001).

Since the adoptive transfer of lymph node and splenic T cells from L. donovani-infected BALB/c to naïve syngenic recipients resulted in enhanced myelopoiesis [10], the culture supernatants of these T cells were tested for their mast cell inducing activity in vitro. It was observed that lymph node T cells were more efficient in eliciting mast cells than the splenic T cells at any stage of infection (Figs 2b; P < 0·001) and that the BM from 120-day infected mice yielded the highest number of mast cells, indicating a rise in MCCP in BM during infection (Fig. 2b). Neutralization of the 120-day postinfection LN T cell supernatant by anti-IL-3 antibody, as IL-3 was known for its mast cell inducing activity [11], reduced mast cell yield by 9-fold suggesting that IL-3 was a major mast cell inducer (Fig. 2c). Indeed, the number of IL-3 secreting T cells increased in Leishmania-infected BALB/c but not in C57BL/6 mice [15]. These observations indicated that MCCP increased in both bone marrow and spleen during Leishmania infection. Similar IL-3-dependent increase in MCCP was reported in Nippostrongylus brasiliensis-infected mice [16]. Therefore, our data suggest that a similar mechanism to augment MCCP is operative in L. donovani infection. In contrast, the low MCCP in a resistant mouse is perhaps due to low levels of IL-3 or less IL-3 responsiveness.

Mast cells release IL-3 and IL-4 to render macrophages susceptible to Leishmaniainfection in vitro

Although Leishmania can infect mast cells [17] (Fig. 3a), it lives primarily in macrophages. Therefore, we investigated whether or not the splenic mast cells modulate the IFN-γ-mediated leishmanicidal activity of macrophages. Pre-incubation of macrophages with the BALB/c-derived mast cell supernatant increased the number of amastigotes per 100 macrophages but neutralization of IL-3 and IL-4 in the supernatant reduced the parasite load significantly (P < 0·001; Fig. 3b,c). However, the BALB/c-derived mast cell supernatants alone did not enhance the parasite load in BALB/c macrophages significantly (data not shown). In contrast, C57BL/6-derived mast cell supernatants restricted L. donovani growth in BALB/c macrophages (Fig. 3d) perhaps due to the presence of significantly higher TNF-α in the C57Bl/6-derived mast cell supernatant (Fig. 3e). The observations suggest that mast cell-derived cytokines play a pro-parasitic role in a susceptible but an antiparasitic role in a resistant mouse. This observation is in accordance with the previous report that IgE-sensitized mast cells release TNF-α, a host-protective cytokine, or IL-4, a disease-promoting cytokine, among a variety of other mediators like histamine [17–21] and that IL-3 acts as a susceptibility factor [15,22]. Since Leishmania infection results in much higher serum IgE levels in susceptible mice than in resistant mice [23], it is possible that mast cells play important roles in Leishmania infection, particularly in a susceptible host by IgE-mediated degranulation [24]. Alternatively, the observed difference in the mast cell-induced leishmanicidal activity suggests that the mast cell function to promote or prohibit Leishmania growth in BALB/c and C57BL/6 mice, respectively, is genetically regulated.

Fig. 3.

Supernatants of mast cells from BALB/c and C7BL/6 mice differentially regulate Leishmania growth in macrophages. (a) Amastigote (A) in mast cells (M) in vivo is shown in the electron micrograph. Mast cell granules (mg) and nucleus (N) are also seen. (b) The culture supernatants of splenic T cells (D10 sup) isolated from BALB/c mice 10 days after L. donovani infection eliminate amastigotes in an IFN-γ-dependent manner. The data presented is mean ± SD (*P < 0·001) (c) BALB/c mast cell supernatants promote Leishmania infection. BALB/c-derived peritoneal macrophages were preincubated with the mast cell supernatants (MC:Sup), generated by culturing the cells with leishmanial antigens (CSA) and day-120 postinfection sera from BALB/c mice. The macrophages were infected with L. donovani promastigotes at a macrophage: promastigote ratio of 1 : 10, cultured for 72 hours in presence or absence of D10 sup as indicated, followed by fixation, Giemsa-staining and counting the number of amastigotes/100 macrophages under a light microscope. In order to detect the inhibitory factor(s) in the mast cell supernatants, the mast cell supernatants were neutralized with anti-IL-3 and anti-IL-4 antibodies. The result, shown as mean ± SD (*P < 0·001), is from a representative experiment that was performed three times. (d) C57BL/6 mast cell supernatants reduce Leishmania infection in BALB/c macrophages. The data presented is mean ± SD (*P < 0·001) and is a representative of one of three individual experiments. (e). BALB/c and C57BL/6-derived mast cells, uninfected (□) or L.donovani-infected (▪), are cultured for eight hours. The culture supernatants were assayed for TNF-α content by an ELISA kit (PharMingen, San Diego, CA, USA). The data presented is mean ± SD (**P < 0·005).

Our data reveals a novel aspect of the host–pathogen interaction where mast cells play a pro-parasitic role in a susceptible host but an antiparasitic in a resistant host. The parasite induces an early mast cell infiltration in a susceptible mouse whereas avoids the same in a resistant mouse, exemplifying the parasite's crucial modus operandi, which is deviation of the host-protective immune response, to ensure its survival. In a resistant host, mast cells kill the parasite early after infection whereas in a susceptible host unable to kill the parasite, the mast cells regulate fibrosis and granuloma formation in liver and bone marrow [16,25], a strategy to contain the parasite, restricting its dissemination.