Immunophenotyping of peripheral blood mononuclear cells and intracellular detection of IL-2 , IFN γ and IL-4 by flow cytometry in patients with actinomycetoma by Nocardia brasiliensis and Actinomadura madurae . Findings in six patients

Mycetoma is a chronic, granulomatous, progressive inflammatory disease; it usually involves the subcutaneous tissue after a traumatic inoculation of the causative organism. Mycetoma may be caused by true fungi or bacteria (actinomycetes) and hence it is classified as eumycetoma and actinomycetoma respectively. Mycetoma immunological studies have frequently addressed humoral aspects. Few reports have addressed the role of cellular immunity in humans, for this reason, we were interested in finding differences in the circulating population of mononuclear cells or in the ability of this pathology to produce cytokines after mitogen stimulation. In this study, immunophenotyping of peripheral blood mononuclear cells (PBMN) and intracellular detection by flow cytometry of IL-2, IFNγ and IL-4 in patients with actinomycetoma by Nocardia brasiliensis and Actinomadura madurae were evaluated. We investigated the expression of T-cells (CD3, CD4 and CD8), B-cells (CD19), monocytes (CD14), and natural killer cells (CD16 and CD56) markers. CD69 and CD25 were used to monitor individual activated cell subsets. The percentage values of the cells were calculated. Our results indicated that PBMC from patients with mycetoma show similar percentages of circulating cells when compared with healthy donors. The expression of IL-2 receptor (IL-2R) by mitogen activation was similar in these two groups. These findings suggest that circulating lymphocytes are not affected by this pathology. Intracellular IL-4 was increased only in patients with mycetoma by N. brasiliensis, suggesting a TH2 profile; this observation has been reported by other authors.


INTRODUCTION
Mycetoma is a chronic, granulomatous, progressive inflammatory disease; it usually involves the subcutaneous tissue after a traumatic inoculation of the causa-tive organism.Mycetoma may be caused by true fungi or by bacteria (actinomycetes) and hence it is classified as eumycetoma and actinomycetoma respectively [1,2].Tumefaction and formation of sinus tracts characterize mycetoma.The sinuses usually discharge purulent and seropurulent exudate containing microcolonies called grains.It may spread involving skin and the deep structures, resulting in destruction, deformity, and loss of function.In rare occassions it may be fatal [3,4].The clinical presentation of mycetoma is almost identical, regardless of the causal organism.However, the progress is faster in actinomycetoma comparatively with eumycetoma.In eumycetoma, the lesion grows slowly, with clear defined margins and it remains encapsulated for a long period, whereas, in actinomycetoma the lesion is more inflammatory, more destructive, and it invades the bone at an earlier period [1,4].Mycetoma lesion has a distinct appearance in a cytology smear which is characterized by the presence of inflammatory cells consisting of a mixture of neutrophils, lymphocytes, plasma cells, histiocytes, macrophages, and foreign body giant cells in grains [5].In tissue sections, three types of tissue reactions have been described (I, II and III).Type I is characterized by actinomycetoma lesion [6,7] where the grain is closely surrounded by polymorphonuclear leukocytes that are the predominant cell type in the inflammatory infiltrate in the skin lesions and occasionally infiltrated by neutrophils, causing its fragmentation.Outside the neutrophil zone, monocytic cells and giant cells are observed.The lymphocytes are scant where the main population is TCD4 + [8].
Cenci et al. [9] demonstrated the cellular elements in the inflammatory infiltrate in skin lesions of actinomycetoma and eumycetoma and suggested that cellular mediated immunity may play a role in mycetoma pathogenesis, in which a marked reduction of Langerhans cells may reflect a depressed cell immune response, partially explaining a chronic condition and unresponsiveness to the treatment.
Studies on the immunopathologic aspects of tissue reaction are much less frequent, immunological studies of mycetoma have frequently addressed humoral aspects [10][11][12][13].Few reports have addressed the role of cellular immunity in humans.The main studies regarding cellular immune response by delayed hypersensitivity to antigens from Nocardia was first describe in 1972 [14] and protective effect after spleen cell transfer from guinea pigs infected with Nocardia asteroids was demonstrated five years later [15].A defective T-cell mediated response in eumycetoma patients was suggested as an important element in this pathology when the lymphocyte proliferation stimulated with phytohemagglutinin affected skin reactivity to dinitrochlorobenzene in these patients [16]; in contrast, infections by Nocardia asteroides did not show these efects [17].
Cell-mediated immunity plays a major protection role against intracellular microbes.Nocardia brasiliensis is a facultative intracellular pathogen that grows in macrophages but not in PMN leukocytes [18].The mechanisms that allow explaining the evasion, resistance, or neutralization of bactericidal action of macrophages and neutrophils are: 1) production of high levels of catalase and superoxide dismutase that reduce the oxygen toxic products generated by phagocyte, 2) reduction of lysosome enzymatic activity of some macrophage populations, 3) blockade of phagosome acidification, and 4) inhibition of phagosome-lysosome fusion [19].
Regarding Actinomadura madurae infections, the ability of this bacteria to persist inside murine macrophages in experimental in vivo and in vitro infections has been reported [20]; as with nocardia infections, this could explain the tendency to chronicity by actinomycetales due their replicative capacity inside the phagocytes, probably this fact is related to blood dissemination as it was demonstrated in experimental mycetoma by Nocardia brasiliensis [21].
In mycetoma, the local host response is characterized by neutrophil chemotaxis and small vessel congestion.Initially, the response is nonspecific, later, macrophages and monocytes present at the infection site are activated by interferon gamma and tumor necrosis factor-alpha; these cells have enhanced microbicidal activity [22].For these reasons, the production of pro-inflammatory and anti-inflammatory cytokines and the expression of their receptors, after stimulation are important indicators of mononuclear cell activation in this pathology.

Patients
Mononuclear cells from four patients with mycetoma by Nocardia brasiliensis, two patients with mycetoma by Actinomadura madurae, and five healthy subjects were evaluated.These patients were diagnosed and treated by the Mycology Service of the Dermatologic Center "Ladislao de la Pascua" in Mexico City.This study was carried out with the written consent of all patients and it was approved by the Ethics Committees of each participating institution.

Intracellular Cytokine Detection: IFN-γ, IL-2 and IL-4 [24]
The production of cytokines was measured through intracellular staining.Briefly, capped polystyrene Falcon tubes (Becton Dickinson) were used to incubate whole blood samples with 25 ng/ml phorbol myristate acetate (PMA) (Sigma), 1 µg/ml ionomycin (Sigma), and 10 µg/ml brefeldin A (BFA) (Sigma) or BFA only (control), for 5 h at 37˚C and 5% CO 2 .Anti-CD3 peridinin chlorophyll protein (PerCP) (Becton Dickinson) was added to aliquots of the stimulated and non-stimulated blood and then incubated for 15 min at room temperature in the dark.After incubation, erythrocytes were lysed with FACS lysing solution (Becton Dickinson) and the samples were incubated for another 10 min at room temperature in the dark.The cells were then centrifuged for 5 min at 500 × g and supernatants were aspirated without disturbing the pellets.FACS permeabilizing solution (Becton Dickinson) was added to the pellets and incubated for 10 min at room temperature in the dark; cell suspensions were then washed with phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) (Research Organics, Cleveland, OH) and 0.01% sodium azide (PBA), and centrifuged for 5 min at 500 × G, the supernatants were removed.For intracellular staining, anti-cytokine monoclonal antibodies (mAbs) were added to the pellets and cell suspensions were incubated at room temperature for 30 min in the dark, ac-cording to the following protocol: FastImmune R INF-γ fluorescein isothiocyanate (FITC)/IL-4 phycoerythrin (PE) (Becton Dickinson) or FastImmune R anti-HuIL-2 FITC (Becton Dickinson), and anti-CD69PE (Becton Dickinson) were added to the pellets previously stained with anti-CD3PerCP.Simultest γ 2a FITC/γ 1 PE with anti-CD3 PerCP was used as isotype control.
For all conditions, the expression of CD69 was measured as an activation control.The samples were washed with PBA and fixed with PBS containing 1% paraformaldehyde (PFA).A fluorescent activated cell sorter FACSCalibur R (Becton Dickinson) equipped with a 15 mW argon ion laser and filter settings for FITC (530 nm), PE (585 nm) and PerCP(Becton Dickinson), emitting in the deep red (> 650 nm) was used.Cells (10,000) were computed in list mode and analyzed gating on CD3 (lymphocytes) using the CellQuest R software.

IL-2ΑR Expression Analysis
A kinetic analysis for the expression of CD25 + (IL-2αR) was made.PBMC from healthy subjects were stimulated with 20 µg/mL PHA for 0, 24, 48 and 72 h and stained for CD25 (IL-2αR).Percentages of lymphocytes positive for CD25 were mesured at each time by  flow cytometry.As it can be seen in Figure 1, maximum expression of IL-2αR (58.66%CD25 + ) was detected after 72 h of activation; however, at this time, the percentage of cell death was also high (60% of cells were positive for propidium iodide staining, data no shown).For this reason, all determinations were done after 24 h of stimulation.

Discussion
Although mycetoma was first described more than 3,000 years ago, the studies on physiopathology began only some decades ago.Many aspects about the immune response during the evolution of this disease are still unknown.A basic question related to the nature of the immunological response in this pathology could be related to the existence of differences in the circulating mononuclear cells in these patients.In our analysis, peripheral blood monocytes, CD4 + and CD8 + T lymphocytes and natural killer cells CD56 + were present with similar values in patients as well as control subjects (Table 1).This fact suggests that the immune response is developed locally and the patients that suffer this chronical disease do not present changes in circulating mononuclear cells (Figure 2).
Host immune response is present in both, human (natural infection) and experimental infections (animal models or in vitro) with Nocardia brasiliensis and Actinomadura madurae.The response is characterized by an intense infiltration of polymorphonuclear cells, being this a constant finding during the entire active infection [26].Infections by Nocardia have been associated with: a) inhibition of the bacteria by monocytes and polymerphonuclear leukocytes, b) activation of macrophages and 3) specific cytotoxicity from a T cell subset [27].However,in the long run, Nocardia depends on the metabolic state of macrophage and neutrophil, the concentration and type of lysosomal enzymes inside the cell and the type of strain of Nocardia [17] and also, probably, the presence of L forms of bacteria inside cells involved in tissue process [28].A deficiency of one of these factors could augment the host's subseptibility to acquire the illness produced by this bacterium.The ideal candidates to modulate these functions are the cytokines which are proteins that regulate the origin, proliferation and activation of the immune cellular response.
There is a wide range of disease severeness for many human pathologic conditions; it is likely to be a spectrum of cytokine production, imbalance, or cytokine dysregulation, at different stages of the disease.In infections like mycetoma, IFN-γ may induce or activate bactericidal mechanisms of infected macrophages and clear the infection.It may induce TH1 lymphocyte proliferation, activate macrophages and reduce the production of other interleukins such as IL-4 and IL-10.In an experimental actinomycetoma model in mice, it was demonstrated that circulating IFN-γ levels increased 10 times the basal levels during the 4 days of infection and decreased with the progression of the disease [29].
The identification of a specific cell surface marker for type 1 or type 2 cytokine-producing cells or the ability to stain intracellulary such cytokines would greatly facilitate the study of these cells and their role in human diseases.Direct intracellular detection of cytokines by flow cytometry has been accomplished, in some cases in combination with cell surface marker expression [30].In this work, intracellular cytokines produced by stimulated T lymphocytes from patients with mycetoma showed an adequate response to activation.Similar percentages of IFN-γ producing cells were detected in both healthy donors and patients.These results demonstrate that cellular immune response of patients is not affected by the pa-thology, at least in these cytokines, as shown in Figure 3.
Instead the cytokine quantification, the study of receptor activity may be a more suitable indicator of cellular activation ability.The measurement of functional receptors by the binding of labeled cytokine has been reported [25]; in mycetoma patients, the expression of IL-2 receptor (CD25) by PHA stimulation showed an increase in patients, this finding demonstrates that patient's CD3 + CD4 + CD25 + cells are able to bind to IL-2 and consequently to be stimulated for proliferation and activation (Table 2).
There is an inverse relationship between cell-mediated (CMI) and humoral immunity in response to antigenic stimuli.TH1 cells are recongnized to provide a better helper activity for CMI whereas TH2 cells are more important for B-cell development and antibody production.Human illnesses regarding type 1 and type 2 cytokine imbalances may explain part of the pathology in this disease; thus restoring the balance may ameliorate the disease [31].The profile of TH1-TH2 responses in mycetoma is unknown.
TH2 cytokines also induce IgE production and eosinophilia.These responses are characteristic of helminth infections, atopic disorders, and uncontrolled infections with intracellular pathogens (e.g.visceral leishmaniasis and lepromatous leprosy) [32].
In this study, T-lymphocytes producing IL-4 were higher only in the patients with N. brasiliensis infection; in contrast, the cells from A. madurae patients were normal.This fact probably indicates that TH2 profile is present in actinomycetoma Nocardia patients.This type of immune response may favor the disease progression.These observations are in agreement with the study of El Hassan [33] who demonstrated the TH2 cytokine pattern in lymph nodes from patients suffering mycetoma by Streptomyces somaliensis, as well as the cytokine production analysis in patients with actinomycetoma caused by Nocardia brasiliensis [34] However, further studies in actinomycetoma are needed to determine the production of other cytokines (TNFα, IL-10).

Figure 1 .
Figure 1.Percent of expression of IL-2αR at different hours of stimulation.

Figure 2 .
Figure 2. Percentage from PBMC evaluated by flow cytometry from mononuclear peripheral blood from healthy volunteers (a) and (d); with mycetoma by Nocardia brasiliensis patients (b) and (e); and Actinomadura madurae patients (c) and (f).PBMC were stimulated with 20 µg/mL phytohaemaglutinin in RPMI, for 24 h at 37˚C and 5 % CO 2, then the cells were stained with: anti-CD25PE, anti-CD4FITC, anti-CD8PE and anti-CD3PerCP.Percentages of T lymphocytes (gated on CD3 + ) expressing CD25 were measured by flow cytometry.

Table 3 .
Percentage of lymphocytes expressing CD69 and producing cytokines upon stimulation.