Evaluation of the phagocytic activity of peripheral blood monocytes of patients with Jorge Lobo ’ s disease

Studies on host-parasite interaction in Jorge Lobo’s disease are scarce, with no report in the literature on the phagocytosis of Lacazia loboi by phagocytic mononuclear cells. Thus, the objective of the present study was to assess the phagocytic activity of blood monocytes in the presence of L. loboi in patients with the disease and in healthy subjects (controls) over 3 and 24 hours of incubation. Statistical analyses of the results showed no significant difference in percent phagocytosis of the fungus between patient and control monocytes. With respect to incubation time, however, there was a significant difference, in that percent phagocytosis was higher at 3 hours than at 24 hours (p <0.01). These results suggest that monocytes from patients with the mycosis are able to phagocyte the fungus, as also observed in control individuals. Key-words: Jorge Lobo’s disease. Phagocytosis. Monocyte. Lacazia loboi.

Jorge Lobo's disease is a cutaneous-subcutaneous mycosis with a chronic evolution caused by the fungus Lacazia loboi (L.loboi) 8 18 .The mycosis predominantly occurs in Brazil but cases have been reported, in decreasing order, in Colombia, Surinam, French Guyana, Venezuela, Panama, Costa Rica, Peru, Ecuador, Bolivia, Mexico, and Europe 12 .According to Opromolla et al 11 , the estimated number of cases of the disease is 458, 295 and those that occurred in Brazil (295 cases) were predominantly in the Amazon region.Jorge Lobo's mycosis mainly attacks people from forest areas close to rivers and flood land with a warm and humid climate, as is the case for workers tapping rubber trees in the Amazon region, and is markedly more frequent among males 6 .The disease has been detected in white, black and Indigenous individuals, with no evidence of greater susceptibility to infection in a given ethnic group 7 .
Clinically, the mycosis is characterized by cutaneous lesions of with a cheloid-like, wart-like infiltrative, ulcerous or gummy aspect, with the possible occurrence of more than one type of lesion in the same patient 16 .Ulcerated lesions seem to be a result of traumatic injuries more than to primary spontaneous ulceration and therefore are clinically secondary 11 .
Histopathologically, Jorge Lobo's disease is characterized by an intense diffuse histiocytic reaction containing large numbers of foreign body giant cells and Langhans' cells, and numerous intra or extracellular parasites.Small numbers of lymphocytes, plasmocytes and neutrophils are also present 7 10 .
Ultrastructural studies of the cutaneous lesions have revealed that, after the fungus is endocytosed by the phagocytic cell and suffers the action of lysosomal enzymes, remnants of the cell wall of L. loboi can be detected in the cytoplasm.These cell wall remnants progressively accumulate inside the macrophages, conferring a foamy aspect, as observed by light microscopy 14 15 .
Macrophages are cells commonly associated with the defense mechanisms of the host against invading microorganisms.They belong to the mononuclear phagocytic system and, in addition to defending the host through phagocytosis and cell digestion, they also interact with and stimulate lymphocytes to participate in the defense mechanisms, thus playing a fundamental role both in nonspecific mechanisms of defense and in the specific immune response 1 .
Few studies on the host-parasite interaction are available for Jorge Lobo's disease and no study exists about the phagocytosis of L. loboi by mononuclear phagocytic cells.However, this topic has been studied in other mycoses such as paracoccidioidomycosis 3 4 and candidiasis 13 , demonstrating the important role of macrophages in host defense.Thus, the aim of the present study was to assess the phagocytosis of the fungus L. loboi by peripheral blood monocyte of patients with Jorge Lobo's disease.

PATIENTS AND METHODS
Patients.The study was conducted on 10 patients with Jorge Lobo's disease from the State of Acre and examined by the clinical staff of the Lauro de Souza Lima Institute, in Bauru-SP, with a diagnosis of the mycosis confirmed by anatomicopathological examination.Fifteen healthy adults, employees of the Lauro de Souza Lima Institute, with similar age to that of the patients, were used as control group.
Blood collection.Twenty ml of venous blood was collected from each patient and control subject with 2 Vacutainer (BD, Minas Gerais, Brazil) tubes containing heparin as an anticoagulant.
Mononuclear cell preparation.Mononuclear cells were separated on a Ficoll-Hypaque density gradient 2 (Sigma, Missouri, USA).The cells were resuspended in 1ml RPMI medium containing L-glutamine and 15mM HEPES buffer (Gibco BRL, São Paulo, Brazil) supplemented with penicillin (100UI/ml)-streptomycin (100µg/ml) (Gibco BRL).Monocytes were counted in a Neubauer chamber by 1:8 dilution of the cell suspension with 0.02% neutral red dye in saline solution.The preparation was left to stand at 37 o C for 20 min and the final concentration of the suspension was adjusted to 5 x 10 5 monocytes/ml.
Monocyte culture.One ml of the monocyte suspension was distributed in duplicate among the wells of flat-bottomed 24-well tissue culture plates (Corning, New York, USA) containing a previously sterilized coverslip measuring 13mm in diameter (Glasstécnica, São Paulo, Brazil).After 2 hours, the supernatants were removed and each well was washed with RPMI medium to remove non-adherent cells.Next, 1ml of RPMI medium supplemented with 15% fresh AB + pooled serum (obtained by combining sera from 5 healthy donors under conditions preserving complement activity) and with antibiotics was added.A 100ml aliquot of the L. loboi suspension containing 5 x 10 5 fungi at the proportion of 1 cell for 1 fungus was added to each well.
The Lacazia loboi suspension was obtained from skin lesions previously collected by surgical removal and stored frozen.The biopsies were processed in 0.85% saline solution and the fungal suspension obtained was filtered through gauze for the removal of tissue remnants and then autoclaved.The fungi present in the suspension were counted in a Neubauer chamber.
The plate was incubated at 37 o C in a 5% CO 2 atmosphere for 3 and 24 hours.After these incubation periods, the coverslips were removed, washed with 0.85% saline and stained with Giemsa.
Evaluation of phagocytic activity.Two hundred monocytes were examined in coverslips at 1000x magnification for enumeration of the cells containing fungus or not.
Statistical analyses.Two-way analyses of variance was used to determine the significance of the difference in percent fungus phagocytosis, comparing patient and control monocytes 19 .The same analyses was also used to determine the effect of time on the percentage of phagocytosis regardless of the group studied.In all circumstances, the level of significance was set at p < 0.01.

RESULTS
The percentages of peripheral blood mononuclear cells phagocyting L. loboi from patients and controls are presented in Table 1.Statistical analyses of the results showed no significant difference in percent phagocytosis by the monocytes of patients and controls.However, there was a significant difference between the time points evaluated, with a higher percentage of phagocytosis at 3 hours than at 24 hours (p <0.01).Figure 1 illustrates L. loboi phagocytosis by peripheral blood monocytes from a patient with Jorge Lobo's mycosis.
We had also the opportunity to assess the phagocytic index of two patients with Jorge Lobo's disease by adding fresh and inactivated serum of the patient himself to the cultures.We observed a higher percentage of phagocytosis with not inactivated serum (33%) than with inactivated serum (24%).

DISCUSSION
The phagocytosis of microorganisms represents one of the nonspecific defense mechanisms of vital importance for the host.The monocyte/macrophage cell lines, commonly referred to as professional phagocytes, can neutralize, engulf and destroy particles, including infectious agents, thus presenting a high phagocytic potential 1 .In this respect, these cells have been frequently evaluated for phagocytic and lytic capacity against pathogenic microorganisms.
In the present study, the evaluation of the phagocytic capacity of blood monocytes from patients with Jorge Lobo's disease revealed that the monocytes of both patients and healthy individuals (controls) presented a similar percentage of phagocytosis, with no statistically significant difference between them.
A survey of the literature did not locate any report concerning the phagocytosis of L. loboi by monocytes of patients with the mycosis, although this topic has been investigated for other mycoses.In paracoccidioidomycosis, a study on the phagocytosis of P. brasiliensis by mouse alveolar macrophages revealed that 64.2% of the macrophages phagocytized the fungus.When the cultures were treated with interferon gamma (INF-γ ) there was no significant difference in percent phagocytosis over a period of 4 hours, but the percentage increased significantly after 24 hours.Similarly, the fungicidal capacity of activated macrophages was also higher after 24 hours 3 .
In a later study, Brummer et al 4 evaluated the phagocytic and fungicidal potential of murine peritoneal macrophages activated or not with INF-γ and observed that after 4 hours 92% of the fungi had been phagocytized by macrophages regardless of whether or not they had been activated.However, only activated macrophages were able to fully eliminate P. brasiliensis within a period of 48 hours.According to the cited investigators, activated macrophages play a decisive role in the resistance to P. brasiliensis.More recently, Moscardi-Bacchi et al 9 evaluated the phagocytosis of blood monocytes from patients with paracoccidioidomycosis and detected a phagocytic rate of 80%.They also observed that monocytes activated with INF-γ markedly inhibited the multiplication of P. brasiliensis.
In the present study, the phagocytosis of L. loboi was assessed at 3 and 24 hours and statistical analyses of the results showed that, regardless of the group studied, phagocytosis of the fungus was higher at 3 hours than at 24 hours.These results are similar to those obtained by Brummer et al 4 who detected 92.7% phagocytosis of P. brasiliensis by mouse peritoneal macrophages at 4 hours, 61% at 24 hours, and 10.2% at 48 hours.
In the literature, most studies on fungus phagocytosis used periods of time ranging from half an hour to 4 hours 3 5 13 17 .Thus, in the present study we opted for the evaluation of phagocytosis after 3 hours and then at 24 hours.
The design of the present study took into consideration what the ideal proportion of phagocytic cell:fungi would be for the cultures.Literature data have revealed wide variations 5 13 17 and therefore we opted for the use of 1 cell:1 fungus.In this respect, Sawyer 13 assessed the phagocytic activity of mouse alveolar macrophages against C. albicans using 1:1 and 5:1 phagocytic cell:fungus proportions and their results showed that the rates of phagocytosis were higher when the 1 cell:1 fungus proportion was used.
In another study, Gaziri et al 5 used the proportions of 1:3, 1:6 and 1:9 cell:fungus for the evaluation of C. albicans phagocytosis by mouse peritoneal macrophages and obtained better results when the 1:9 proportion was used.These investigators demonstrated the important role of the complement system in the opsonization of C. albicans and later phagocytosis by macrophages.They showed that phagocytic activity was much higher when fresh serum was added to the cultures as a source of complement.When fresh serum was added, phagocytosis was 50.6% and when heat-inactivated serum was used, phagocytosis was 5.4% using the proportion of 1 cell:3 fungi.
In the present study, monocyte cultures contained fresh AB + serum as a source of complement.As also done by Gaziri et al 5 , we had the opportunity to assess the phagocytic index of two patients with Jorge Lobo's disease by adding fresh and inactivated serum of the patient himself to the cultures.The results obtained suggest that the antibody and complement favor the opsonization and phagocytosis of the fungus.However, a larger number of patients is necessary for a better study of the participation of these opsonins in L. loboi phagocytosis.
Whatever, the results reported here represent a first assay for the evaluation of one of the nonspecific defense mechanisms of the host and reveal that the monocytes of patients with Jorge Lobo's disease are able to phagocyte L. loboi, there was no significant difference between patients and controls.They also show that the rate of fungal phagocytosis was higher during the period of 3 hours compared to 24 hours, suggesting that during the later period the phagocytes may be involved in the process of lysis of the phagocytized fungus.