| Objective:Using rabbit skin liquefaction model to assess the effects of immunomodulators including recombinant human interferon-γ (rIFN-γ), recombinant human interleukin-2(rIL-2), dexamethasone and cyclophosphamide (CTX) on the development of tuberculosis (TB) lesions and tubercle bacillus load in liquefied caseum, establish essential method for therapeutic agent evaluation and study the mechanisms of tuberculosis liquefaction.Methods:Animals were divided into six groups with four rabbits in each group. They were intramuscularly injected, respectively, with recombinant human interferon-y (rIFN-γ), recombinant human interleukin-2(rIL-2), a low-dose dexamethasone, a high-dose dexamethasone or cyclophosphamide (CTX) for8weeks, or with the same volume of saline as the control. The injections were performed daily. Seventeen days post-administration with each immunomodulator, the rabbits were injected intradermally with5×106CFU of BCG in0.1ml saline into two sites on each flank. The lesions were checked daily and the pathomorphologic features of the lesions were examined at a certain stage. And we recorded the time when the lesions formed granulomas, when the granuloma peaked in size, when liquefaction was first detected, when liquefaction seemed maximum, when the skin above the granuloma ulcerated, when healing began, and when the lesions seemed to be healed. The number of tubercle bacilli within liquefied caseum from skin lesions was determined at the peak of liquefaction. Rabbit serum cytokine levels from each immunomodulator group were determined by ELISA at six stages of the development of lesions. The correlations in the serum levels of IL-2and the bacillary load in liquefied caseum and the development of tuberculosis liquefaction were analyzed.Results:1. The effects of different immunomodulators on the BCG-induced rabbit skin lesions were different. For example, the time of granuloma, liquefaction, ulceration and healing in different groups were different, the volume of skin lesions at different stages were different.In the rIL-2group, the BCG-induced skin lesions appeared more quickly and reached the ulceration and the peak of liquefaction earlier than the other groups. The beginning of the liquefaction appeared at the8th day post-infection with BCG. The beginning of the ulceration appeared at the11th day post-infection with BCG. There were two peak of liquefaction in this group. The first peak of liquefaction appeared at the13th day post-infection. The volume of the liquefied lesions in the group was larger than the others, the lesion volume at the first peak of liquefaction was3289±354mm3, was significant larger than the volume of control group (2563±17mm3)(P<0.05). The lesion subsided more rapidly than that of the other groups, the beginning of first healing appeared at the17th day post-infection.The rIFN-γ group followed with the rIL-2group. There were also two peak of liquefaction occurred in this group. The development period of skin lesion in the CTX group was similar to that in the control group, but the volume of the lesions was smaller than that in the control group.The lesion in the low-dose dexamethasone (DL) group was the smallest among all groups. The volume of lesion at the granuloma stage was751±153mm3, was significant smaller that in the control group (1699±149mm3)(P<0.05). The volume of lesion at the peak of liquefaction was601±97mm3, was also significant smaller than that in the control group (2563±17mm3)(P<0.05). And the liquefaction in this group was not obvious. In the high-dose dexamethasone (DH) group, the granuloma was not obvious, and the liquefaction of the lesions had not been observed.2. The number of tubercle bacilli within liquefied caseum at the peak of liquefaction stage in different groups was different. The CFU in the rIL-2group was the lowest in all groups. The CFU from the first peak of liquefaction in the rIL-2group (3.0×106CFU/g) were significantly lower than the CFU in the control group (6.9×106CFU/g). The CFU in the second peak of liquefaction of the rIL-2group (1.7×105CFU/g) was significant lower than the first peak. In contrast, the CFU of group DL (2.8×107CFU/g) and group CTX (1.5×107CFU/g) were significantly higher than the control group, and the bacterial load in the DL group was the highest in all groups.3. The serum levels of rabbit IFN-γ, IL-2, IL-4and IL-17at different stage of lesions in different treatment groups were different.In the rIFN-γ treated group, the serum IFN-y level increased while granuloma (1706.3±104.3pg/ml) and liquefaction (2662.9±208.5pg/ml) developed, they were higher than the level at pre-administration (866.2±29pg/ml). The level reached to the highest at the peak of liquefaction stage (2662.9±208.5pg/ml), and the level was higher than that at the same stage in the control group (964.9±51.7pg/ml).The serum IL-2levels increased with the administration of both rIFN-γand rIL-2during the granuloma stage (rIL-2group:289.1±14.5pg/ml; rIFN-γ group:211.1±17.6pg/ml), the liquefaction stage (rIL-2group:318±25.7pg/ml; rIFN-γ group:297.9±23.2pg/ml) and the healing stage (rIL-2group:205.7±9.9pg/ml; rIFN-γ group:231.6±19.8pg/ml), they were all higher than the level at pre-administration (rIL-2group:103.5±3.9pg/ml; rIFN-γ group:297.9±23.2pg/ml). At the peak of liquefaction stage, the level reached the highest. And the level at the stage in the rIL-2group (318±25.7pg/ml) and the rIFN-γ group (297.9±23.2pg/ml) were higher than that in the control group (201±5.9pg/ml).In the control group, the serum IL-4level increased during liquefaction stage (91.2±10.1pg/ml) and healing phase (87.7±3.1pg/ml), they were high that the level at pre-administration (50.6±0.5pg/ml).The serum IL-17levels increased at the liquefaction stage in all groups. In the rIFN-γ group, the level at the peak of liquefaction (130.1±9.6pg/ml) was significant high than that at pre-administration (54.4±2.3pg/ml).4. The serum IL-2levels at the peak of liquefaction were inversely correlated with number of bacilli present (r2=0.875, P=0.020), and directly correlated with the lesion size (r2=0.748, P=0.058).Conclusion:1. Different immunomodulators made different effects on both the development of tuberculosis lesions (especially the development of liquefaction) and the survival of the mycobacteria within lesions.2. IL-2accelerated the process of tuberculosis liquefaction and enhanced the antibacterial immune response. Dexamethasone inhibited the development of tuberculosis liquefaction, but it facilitated the survival of mycobacterium.3. We have established the basic method for evaluating herapeutic agents on the rabbit skin liquefaction model. |
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