Evaluation of amastigote reactive cells in human cutaneous leishmaniasis caused by Leishmania aethiopica

Abstract

Lymphoproliferative responses to three affinity chromatography purified amastigote antigens of Leishmania pifanoi, P-2, P-4 and P-8, were evaluated in peripheral blood mononuclear cells (PBMC) from patients with Ethiopian cutaneous leishmaniasis. Antigen-stimulated cells were analysed for the percentage of CD4⁺, CD8⁺ and CD16/56⁺ cells and the expressed levels of gamma interferon (IFNγ) and interleukin (IL)-10 were determined in culture supernatants. The amastigote antigens induced cellular responses in leishmaniasis patients with heterologous Leishmania parasite infection. These responses were compared to those of freeze-thawed L. aethiopica promastigote antigen stimulation. The membrane protein (P-8), and to a lesser extent the megasomal/cytoplasmic cysteine proteinase(P-2), induced proliferation with high levels of IFNγ and IL-10 production in cells from patients with active L. aethiopica lesions. CD16/56⁺ NK cells were the main cell types induced to proliferate in response to P-8 and P-2 stimulation, followed by CD8⁺ cell populations. P-4 had no such effect. This contrasts from previous studies of New World human leishmaniasis where P-4 and P-8 were stimulatory. The success of a particular molecule in the induction of a response with a protective phenotype may be dependent on the infecting Leishmania spp. To our knowledge, there are no studies that directly compare the New versus Old World cutaneous leishmaniasis in respect of NK cell and IL-10 responses. Our studies indicate that some leishmanial molecules are recognized across the species, while others are apparently more species specific.

INTRODUCTION

The complexity of the host immunological responses has proven to be arduous in the vaccine development for parasitic infections. In the efforts to develop a vaccine against Leishmania infection both animal models and human research have been pursued. Among the many Leishmania vaccine candidates that have been identified in murine studies, are three affinity chromatography purified amastigote antigens. These molecules, P-2, P-4 and P-8 antigens are stage-specific and are initially derived from in vitro cultivated amastigotes of Leishmania pifanoi[1]. Molecules that are specific to or up-regulated in the amastigote stage are relevant to study since this stage is the progressive form found in the infected mammalian host. Leishmania synthesized cathepsin L-like cysteine proteinases are actively expressed in the amastigote forms and enables the parasite to infect and proliferate efficiently in mammals [2]. The P-2 molecule, which represents 1% of the total L. pifanoi amastigote protein, is a cysteine proteinase primarily associated with the internal megasome organelle although some surface localization of this molecule has also been reported [3]. The P-4 molecules (33–35 kD) are proteins associated with the membrane of the endoplasmic reticulum [4], while the P-8 antigen is a glycolipid-protein complex associated with the external surface membrane of the amastigote [5]. P-4 and P-8 were the major molecules that induced significant cross protection against infection with either L. amazonensis or L. pifanoi in model studies using mice with different H-2 haplotypes. P-2 on the other hand afforded protection in these mice only at low challenge doses [1]. When these molecules were tested in vitro using cells from American cutaneous leishmaniasis patients, P-4 and/or P-8 proved to be the inducers of Th1-like T cell responsiveness [6,7]. Little/no response was found to the P-2 cysteine proteinase. In the present study, we pursued the investigation of immune responses induced by the three molecules in cells of L. aethiopica-infected cutaneous leishmaniasis patients.

Protection against Leishmania infection has been shown to involve CD4⁺[8,9] as well as CD8⁺[10,11] cells with IFNγ involvement. In recent years NK cells have also been shown to have an important role in this protection in Leishmania infected mice, through the capacity of NK cells to secrete IFNγ[12]. Our own studies in human leishmaniasis implicate NK cells in protection against L. aethiopica[13,14]. The search for Leishmania molecules that are able to induce immune-protection with appropriate cellular immune responses has revealed purified [1] and recombinant antigens [15] as well as DNA encoding major Leishmania proteins [9]. In this study, we have evaluated the phenotype of proliferating lymphocytes and cytokine responses of PBMC from Ethiopian cutaneous leishmaniasis patients, due to L. aethiopica (Old World) infection, in response to the purified amastigote proteins (P-2, P-4 and P-8 antigens) that were derived from L. pifanoi, New World species (L. mexicana complex). An appropriate immune response elicited by one species of Leishmania across different species could be an advantage in generating a general vaccine for cutaneous leishmaniasis.

MATERIALS AND METHODS

Patients

Ten leishmaniasis patients attending the ALERT clinic, in Addis Ababa, Ethiopia, consented to give blood for this study after the nature of the study had been explained to them. This project has received local ethical clearance. These patients had active cutaneous lesions due to Leishmania as confirmed by identification of parasites in smears and/or cultures of scrapings from the lesions. All patients except one had single lesions and the duration of the lesions in these patients varied from one month to three years (Table 1). L. aethiopica is the only Leishmania species causing cutaneous leishmaniasis in the catchment area where these patients came from [16]. The patients were tested in the chronological sequence in which they reported to the clinic, but the data is arranged according to the length of infection. The results from one patient with cutaneous L. infantum leishmaniasis [17] are also included in this study for comparison.

Table 1.

Clinical data of cutaneous leishmaniasis patients. Patients were tested in the order in which they attended the clinic (illustrated by the sample ID no.).

Sample code	Sample ID no.	Clinical status	Duration of lesion	Lesion	Age (years)	Sex
LP1	1914/97	LCL	4 weeks	Single	18	Female
LP2	1912/97	LCL	5 weeks	Single	15	Female
LP3	0072/98	LCL	3 months	Single	21	Male
LP4	1904/97	LCL	4 months	Single	25	Male
LP5	1143/98	LCL	6 months	Single
LP6	1842/97	LCL	7 months	Single	29	Female
LP7	1941/97	LCL	8 months	Single	20	Female
LP8	1913/97	LCL	9 months	Single	17	Male
LP9	1915/97	LCL	10 months	Single	19	Male
LP10	1228/98	DCL	3 years	Multiple	22	Male
LIP	6910/97	L. infantum	15 years	Multiple	58	Male

Data presented according to length of infection.

The patients described here have previously been reported in a study testing another leishmaniasis candidate antigen LACK [18].

Antigens and mitogens

The cytoplasmic cysteine proteinase antigen P-2, and membrane proteins P-4 (internal) and P-8 (external) derived from L. pifanoi, were used at a predetermined final concentration of 0·25 µg/ml. Freeze-thawed L. aethiopica promastigote antigen (Ft-Leish) was prepared as previously described [19] and used at an already determined optimal concentration of 1·25 × 10⁶/ml. Purified protein derivative of tuberculin (PPD; Statens Serum Institutet, Copenhagen, Denmark) and the T cell mitogen phytohaemagglutinin (PHA; Murex, Hartford, UK) were each used at a final concentration of 12·5 µg/ml.

Preparation and stimulation of mononuclear cells

Mononuclear cells were separated from defibrinated peripheral blood on a ficoll gradient [20]. Cells were washed and adjusted to the appropriate concentration in RPMI (Gibco BRL, Paisley, UK) containing 2 mm l-glutamine, 100 U penicillin (Gibco), 100 µg/ml streptomycin (Gibco) supplemented with 10% heat inactivated normal Swedish AB serum. Cells were plated and cultured for six days as previously described [14].

Results of proliferation are expressed as counts per minute (CPM) or calculated as Stimulation Indices (SI = CPM of cells in stimulated cultures/CPM of cells in unstimulated cultures). A response was defined as an SI of 2.

Determination of IFNγ and IL-10 levels in supernatants

Supernatants were harvested after 72 h incubation from cells incubated as above, to assess the levels of IFNγ and IL-10 using commercial ELISAs (MABTECH, Stockholm, Sweden) following the manufacturers’ instructions. The sensitivities of the cytokine assays for IFNγ and IL-10 were 27 pg/ml and 9·8 U/ml, respectively.

Phenotype analysis of proliferating cell types

Cell surface markers were examined by FACS analysis as previously described [18]. Phenotype analysis of stained cells was done on a FACScan (Becton Dickinson, Mountain View, CA, USA). An enrichment of large blast cells after six days culture in response to antigenic stimulation was observed in responder MNC. Determined by the forward light scatter, smaller cells with the least light scatter, as seen in most unstimulated cultures, were gated in R1 and the area outside this region was gated R2. In response to antigen stimulation, blast cells undergoing activation were scattered forward-most, away from the R1 cell population into the R2 region. The proportional increase of responding cells of a particular cell type was calculated taking the total number of cells in R2 into consideration using the following formula:

The mathematical correction factor of +1/+1 was necessary since in some instances the percentage of large cells of a particular phenotype in the unstimulated cultures was zero.

Statistical analysis

Students t-test and One Factor anova were used to evaluate the group means and the variances between groups, respectively, after establishing that the data fulfilled the criteria for the use of parametric tests. Correlation coefficient was used to evaluate the relationship of the independent antigen responses.

RESULTS

Proliferative responses

Proliferation of cells in response to amastigote antigens was evaluated and compared with Ft-Leish antigens (Fig. 1). The PBMC from all 10 L. aethiopica-infected patients had high levels of proliferative responses to the homologous crude Ft-Leish Ag stimulation. In addition, the cells from the L. infantum-infected patient also showed strong proliferative responses to the L. aethiopica antigen. Cells from 8 of 10 L. aethiopica infected patients responded to P-2 (mean SI = 26 ± 33), 7 of 10 responded to P-4 (mean SI = 32 ± 51 and cells from all 10 patients were induced to proliferate in response to P-8 (mean SI = 56 ± 77) stimulation.

Fig. 1.

Proliferative responses induced by P-2 (▪), P-4 , and P-8 of the amastigote antigens of L. pifanoi, and Ft-Leish (□) of promastigote antigens of L. aethiopica, in PBMC of patients with cutaneous leishmaniasis. Unstimulated cultures in these patients ranged from 343 to 2908 cpm (mean cpm = 666 ± 368). Results calculated as stimulation indices (SI) are plotted on a logarithmic scale.

The proliferative responses of cells from the L. infantum patient were enhanced after treatment for those antigens to which there was a response prior to treatment. The stimulation indices to P-2 were 1·3 and 0·8, to P-4 were 4·4 and 9·6 and to P-8 were 2·6 and 8·1, before and after treatment, respectively.

PPD and PHA induced proliferation were in the range of SI 5–219 (mean SI = 77 ± 78) and 13–363 (mean SI = 134 ± 117), respectively, in cells of L. aethiopica patients.

Cytokine analysis

Figure 2 shows IFNγ and IL-10 levels measured in 72 h culture supernatants. Ft-Leish induced IFNγ production in the culture supernatants of all 10 L. aethiopica patients with levels ranging from 269 to 5437 pg/ml (mean = 1907 ± 1712). In cells of the L. infantum patient, Ft-Leish induced 299 pg/ml before and 551 pg/ml after treatment (data not shown). P-8 induced high levels of IFNγ ranging from 1116 to 7880 pg/ml (mean = 4264 ± 2221) in cells from all nine LCL patients. P-2, albeit to a lower degree, also induced IFNγ secretion into the culture supernatant in cells of all the nine L. aethiopica patients with single lesions (175–7432 pg/ml; mean = 2647 ± 2245). Measurable IFNγ levels ranging from 410 to 6399 pg/ml (mean = 2208 ± 2317) were induced in response to P-4 in five out of the nine patients. There was no IFNγ production induced in response to any of the amastigote antigens by the cells of the L. aethiopica patient with multiple lesions (LP10).

Fig. 2.

Levels of measurable (a) IFNγ and (b) IL-10 in 72 h culture supernatants in response to P-2 (▪), P-4 , P-8 and Ft-Leish (□) in PBMC of cutaneous leishmaniasis patients.

Ft-Leish induced low IL-10 levels in five of the L. aethiopica patients (range = 9–63 units/ml; mean = 26·6 ± 19) but no response in the L. infantum patient. Of the amastigote antigens the highest levels of IL-10 were measured in response to P-8 stimulation in cells of all L. aethiopica patients tested (range = 26–370 unit/ml; mean = 202 ± 111). P-2 induced IL-10 (range = 26–261 unit/ml; mean = 127 ± 74) in nine of the 10 L. aethiopica patients. Cells from only one patient (LP1) had measurable IL-10 in response to P-4 stimulation (25 units/ml). This individual had no measurable IFNγ to P-4.

Proliferating cell types

The overall total percentage of CD4⁺ cells tended to be frequently higher than CD8⁺ and CD16/56⁺ cells in response to the amastigote as well as the Ft-Leish antigen stimuli (data not shown).

Data were analysed in terms of proportional increase in cell types, taking into consideration the total number of cells in the R2 population (see Materials and methods). The highest proportional increases in response to stimulation with any of the amastigote molecules in the cells from the patients were in the CD16/56⁺ cell populations (Fig. 3). P-8 and to a lesser degree P-2 stimulation induced mainly CD16/56⁺ and CD8⁺ cell responses. P-4 induced moderate proportional increase of CD16/56⁺ cells in 3 single lesion L. aethiopica patients and in general to a much lower extent than that induced to the other amastigote molecules. CD8⁺ cell proliferation to P-4 was appreciably induced in only two of these patients. The patient with multiple lesions, LP10, had no response to P-4. In addition, a proportional increase of above 20% CD16/56⁺ cells to Ft-Leish stimulation was evident for 6 of 10 L aethiopica patients. CD4⁺ cells in unstimulated cultures initially represented a large percentage of the total cells; as a result, the proportional increase in CD4⁺ cells, in general, was low. Only in the PBMC from the L. infantum leishmaniasis patient (after treatment) was there a significant increase in the proportion of CD4⁺ T cells in response to the P-4, and P-8 antigens (data not shown). However, stimulation with Ft-Leish demonstrated a more profound proportional increase in CD4⁺ cells than the stimulation with the amastigote antigens in all L. aethiopica infected patients tested. In fact, in five L. aethiopica patients, the proportional increases in the levels of responding CD4⁺ T cells to Ft-Leish antigen were higher than for the CD8⁺ T cells.

Fig. 3.

Phenotype of responding cells induced to proliferate to (a) P-2, (b) P-4, (c) P-8 and (d) Ft-Leish antigen stimulation in six-day cultures in PBMC of patients with cutaneous leishmaniasis. Data is presented as proportional increases of large cells above the background level using the formula in materials and methods. ▪ CD16/56+, □ CD8+, CD4+.

Correlation between the responses to the tested antigens

The proportional increase of CD16/56⁺ cells in response to P-8 tended to correlate to that of the Ft-Leish stimulation (R = 0·658; P = 0·0388). There were, albeit close, no correlation in the responses of P-2 (R = 0·548; P = 0·101) and none of P-4 (R = 0·159; P = 0·6826) stimulations to the Ft-Leish responses.

The magnitude of the proportional increase of CD8⁺ cells in response to P-4 correlated significantly to Ft-Leish stimulation (R = 0·814; P = 0·0076) while P-2 (R = 0·28; P = 0·4338), and P-8 (R = 0·322; P = 0·3646) responses did not. The P-2, P-4 and P-8 induced CD4⁺ cell proportional increases showed a significant correlation to the response to Ft-Leish stimulation (R = 0·614, P = 0·0592; R = 0·92, P = 0·0004; and R = 0·901; P = 0·0004, respectively). A preferential CD8⁺ cell response was observed in cells from two donors (LP2 and LP7) to stimulation with Ft-Leish and the amastigote antigens.

DISCUSSION

This study assesses the potential of amastigote vaccine candidate molecules derived from L. pifanoi, a New World Leishmania species, to elicit in vitro recognition by PBMC from patients with other forms of cutaneous leishmaniasis, namely that caused by the Old World Leishmania, L. aethiopica. These amastigote proteins, P-2, P-4 and P-8, have been shown to confer varying levels of immune-protection in BALB/c mice [1]. A study using cells from L. braziliensis infected patients with active disease showed that, P-4 and P-8 induced similar percentages of CD4⁺ and CD8⁺ cells to proliferate with Th1 type cytokine profile, in supernatants both before and after cure of disease. The response of patients to the cysteine proteinase molecule, P-2 in this study, however, was minimal [6].

Our results show that cells of cutaneous leishmaniasis patients infected with L. aethiopica could proliferate in response to the L. pifanoi amastigote antigens. The strongest and most consistent response was to P-8 but P-2 and P-4 also induced some proliferation in a portion of the patients examined. The cell types induced to proliferate in response to the P-2 and P-8 molecules were mainly CD16/56⁺ cells and to a lesser extent CD8⁺ cells when the proportional increase was taken into consideration. The response in general, was lower for the P-4 molecule. CD4⁺ cells were not induced to proliferate much above the background level in these PBMCs to any of the amastigote antigens. These results differ somewhat from those of Coutinho et al. [6] and could be a consequence of the difference in the infecting Leishmania species in the patients of the two studies. This is supported by the finding that, CD4 response was evident in response to P-8 and P-4 in the L. infantum patient after treatment. Alternatively, the contrasting finding could be because we have taken into consideration the proportional increase rather than just the percentage of proliferating cells. The latter explanation may not be so important since when cells were stimulated with Ft-Leish antigen prepared from promastigotes of L. aethiopica, CD4⁺ cells were induced to proliferate in 80% of the patients tested. Stimulation indices to this antigen were also higher than to the amastigote antigens at the concentrations tested. It is also important to note that the studies conducted by Coutinho et al. [6] did not assess the responding NK cell phenotype. Unfortunately, in this hospital based study in Ethiopia we were not able to test patients cured of L. aethiopica infection since the patients could not be convinced to return to the hospital after cure. While the data from a single patient is far from conclusive, in the cells of the one patient whose cells could be tested before and after treatment of L. infantum infection, P-4 and P-8 induced significant proliferation and the levels of response tended to be slightly higher after treatment.

We have previously shown that PBMC from non-Leishmania exposed healthy Swedish blood donors could respond to L. aethiopica antigen via proliferation of predominantly CD16/56⁺ cells followed by CD8⁺ cells [13]. Such reactivity was evident in about 30% of those so far tested over the years. Studies in healthy unexposed Swedish blood donors and healthy Ethiopian individuals, showed P-2 to primarily stimulate NK and CD8 cells with the induction of both Th1 and Th2 related cytokines. In the present study, P-2 responses observed (in only some individuals) are most likely independent of Leishmania exposure. Taken together, the results indicate that P-2 seems to induce more of an innate type response rather than an immune recall response.

In cells from patients with cutaneous leishmaniasis from Ethiopia, CD4⁺ cells are the main proliferating phenotype in response to Ft-Leish antigen [13], although in some patients the CD16/56⁺ cells could proliferate in response to L. aethiopica antigen stimulation [14]. A significant CD16/56⁺ response coupled with prominent CD8⁺ cell response to Leishmania stimulation is proposed to be a marker for the course of healing processes [14].

P-4 induced some but low CD16/56⁺ and/or CD8⁺ cell proliferation and no CD4⁺ cells in PBMCs of L. aethiopica patients. There were, however, strong proliferative responses in these patients in response to P-4 stimulation that makes the cellular source of proliferation not immediately obvious. The proliferating PBMC may be accounted for by B cell proliferation, which was not addressed in this study. B cells are capable of producing IL-10 and IL-12, both of which are stimulators of NK cells, which were observed to proliferate in response to P-4. Intracellular cytokine and surface staining by FACS analysis would have allowed the identification of the cellular source of the cytokines, however, these experiments could not be done at the time.

Of the amastigote antigens, P-8 was the strongest inducer of proliferation with IFNγ and IL-10 production in the L. aethiopica patients involving primarily CD16/56⁺ and CD8⁺ cells, but also CD4⁺ cells in some patient cells. This antigen was well recognized by cells of the L. infantum patient by proliferation, mainly of CD4⁺ and CD16/56⁺ cell types, with IFNγ but not IL-10 production. In our earlier studies IL-4 was not found to be a consistent feature of leishmaniasis due to L. aethiopica, whilst an association of IL-10 and disease development has been observed [21]. However, the presence of IFNγ as well as IL-10 in response to the P-8 antigen suggest that the level of IL-10 is insufficient to completely down-regulate IFNγ production and hence, the effects of this cytokine. The individual variation in response to the amastigote antigens in the patient cells could in part be due to the presence of more than one complex of the molecules as has been described for P8 [5].

The basis for a particular molecule to be successful in the induction of a protective response is as yet not clear. While the consensus is that a Th1 response is required for protection, it is unclear what the adequate level of such a response is and what levels could enhance, rather than reduce the symptoms. Thus, the important role of regulatory Th2 cytokines has not been elucidated. In this context, we have shown that proliferation and IFNγ induction is a consequence of different cell types and different leishmanial antigens [22]. It has been proposed, however, that genetic background of the host as well as the infecting Leishmania species could direct the outcome of the immune responses to infection [23]. Of the amastigote antigens of L. pifanoi, P-4 and P-8 have been shown to induce a Th1-like cytokine profile, which is maintained after cure in cells of L. braziliensis infected individuals [6]. In the present study, however, P-8 induced immune responses with a mixed Th1 and Th2-like cytokine profile, in cells of patients with L. aethiopica infection.

If a leishmaniasis vaccine requires the stimulation of CD4⁺ T cells as well as the induction of IFNγ and less of a Th2 cytokine response, then of the molecules tested P-4 does not appear to be an optimal candidate for a vaccine against L. aethiopica. However, if the overall cytokine response is determinant of healing without the involvement of CD4 response, P-4 could be entertained as a vaccine candidate molecule for L. aethiopica infection as the latter two criteria are met. P-8 seems to be consistently active in inducing CD16/56⁺ and CD8⁺ cells, both of which, are associated with resistance and healing processes, in individuals from different parts of the World (Ethiopia and Sweden) and thus probably with different genetic background as well as in the different infecting Leishmania spp. of L. aethiopica and L. infantum. The potential of a vaccine candidate to elicit a balanced IFNγ and IL-10 responses may be beneficial due to the regulation IL-10 may afford in avoiding excessive effects of IFNγ.

In summary, the L. pifanoi amastigote antigens are recognized by patients infected with distinctively different species of Leishmania. Of the three molecules, the membrane proteins P-8 and P-4 could be further explored for vaccine development against cutaneous leishmaniasis (Old World and New World), employing modulatory adjuvants and/or delivery systems that induce a CD4 as well as CD8 Th1-like T cell response.