Suppression of ADCC by Immune Complexes Formed in Vitro in Mycobacterium leprae‐Infected Mice

Antibody‐dependent cellular cytotoxicity (ADCC) was assessed in mice infected experimentally with Mycobacterium leprae and injected simultaneously with in vitro‐formed immune complexes (IC). Significant decrease in the ADCC function was observed in animals given IC at zero day (OdIC) and 3 months (3mIC) post inoculation with M. leprae, when ADCC activity was assessed at 3, 6 and 9 months period. From the data obtained we believe that ADCC is suppressed by IC formed in vitro.

. The role of NK cells in leprosy is uncertain. Cytotoxic/ lytic mechanism may be very important in containing lepra bacilli in leprosy patients. The importance of NK mechanism in the elimination of cells harboring M. leprae is highlighted by the finding of non-T cytotoxic cells influx at purified protein derivative (PPD)-induced delayed-type hypersensitivity (DTH) site in lepromatous leprosy (LL) patients (5). However, immune complexes (IC) formed due to the massive antigenic assault and an equally evoked antibody load could be deleterious to the functioning of the immune system. In an earlier study, we reported that ADCC was largely unaltered in M leprae-infected animals (2). In the present study we have investigated the effect of IC formed in vitro on the ADCC activity in experimental leprosy. From the data obtained we believe that ADCC is suppressed by IC formed in vitro. Abbreviations : ADCC, antibody-dependent cellular cytotoxicity; AFB, acid-fast bacilli; AMLS, anti-Mycobacterium leprae serum; CMI, cell-mediated immunity; DTH, delayed-type hypersensitivity; ENL, erythema nodosum leprosum; FRBC, fowl red blood, cells; IC, immune complexes; NC, normal control; NI, normal infected; NK, natural killer; PPD, purified protein derivative; TRC, thymectomized/irradiated control; TRI, thymectomized/irradiated infected; ZE, zone of equivalence.
the Central Animal Facility of the Institute were employed for the present investigation. The strain has been found to be susceptible to M. leprae infection (13). Thymectomy was carried out at four weeks of age in the thymectomized/irradiated group, and inoculation of M. leprae (as described later on) was carried out a week later. Animals in the normal (unthymectomized) group were also age-matched. The experimental study, divided into two main phases, comprised animals not given IC (Phase I) and those to whom IC were administered (Phase II). The result of the Phase I study has already been published as mentioned above (2). Phase II animals were given in vitro-formed IC either on zero day (OdIC batch) at 3 months (3mIC batch) or at 6 months (6mIC batch) after injection of M. leprae in the infected groups and at a corresponding period in the uninfected control groups. The following groups were thus formed: obtained at 3 (P CO .05) and 6 month (P CO .01) study only and no change was observed at 9 months. In TRC/OdIC batch the mean counts obtained during the three different periods of 3, 6, and 9 months were 58.7+1.9, 54.4+3.5 , and 58.5+5.2, respectively. The decrease was significant (P<0.05) at 3 and 9 months study in the TRI/OdIC batch compared to TRC/OdIC and only at 9 months period (P <0.01) compared to NI/OdIC. 3mIC batch. Significant decrease in values (P 0.01) was evident in NI/3mIC batch at 9 month study period compared to NC/3mIC. No significant difference was seen between any other groups at any period of study. 6mIC batch. No significant change .was seen between any of the group when IC were administered at 6 month period post inoculation with M. leprae.
(b) Acid-Fast Bacilli (AFB) Counts (Table 2) The results of AFB counts have already been published earlier (15). In the NI/OdIC batch, establishment of infection was evident by as early as 3 months in contrast to NI group not adminis-tered immune complexes. A steep increase in the bacillary counts was seen at 6 months which remained static at 9 months. IC administration at 3 months did not bring about any appreciable increase in the bacterial counts compared to NI/ OdIC batch. However, there was a significant increase in the AFB harvest at 9 months period in the 6mIC batch. Enhancement of infection was more evident in the immunosuppressed group (TRI) of animals administered IC.

Discussion
In the ADCC mechanism, non-sensitized effector cells bearing Fc receptors bind to antibody-coated target cells and induce their lysis. The ADCC mechanism was seen to be largely unaltered in the Phase I animals not administered IC with an increase in the cytotoxic activity observed in the lymphocytes of NI mice at the end stage of sacrifice (2).
In the OdIC batch the lysis of target cells by cytotoxic cells was seen to be significantly decreased in both the groups of infected (NI and TRI) animals compaied to uninfected (NC and  TRC) groups, but in the 3mIC batch only in NI animals at 9 month period. Thymectomy and irradiation caused further suppression of ADCC in the TRI/OdIC group at the end of the observation period of 9 months. However, no difference in the suppression of ADCC was seen in the 3mIC and 6mIC batches of the thymectomized irradiated group compared to the non-pretreated groups. This could be due to the enhanced infection occurring as a result of immunosuppressive pretreatment given to this group of animals, which in turn adversely affected the cytotoxic function of the lymphocytes. The possibility of suppression induced by macrophage is very unlikely as only non-adherent cell population was used in the assay. It has been suggested that IC in high concentrations can directly inhibit NK function, while at considerably low concentration, they can deplete some of the effector NK cells through complement lysis (12). Humphres et al (4) observed a decrease in NK cell activity in a group of patients undergoing episode of erythema nodosum leprosum (ENL) reactions which is thought to be an ICmediated process, but not in those with stable lepromatous disease. The decrease in the cytotoxic killing in IC-administered group may also be interpreted on this basis. The cytotoxic cells have Fc receptors and in vivo generation of IC in the infected animals could block some of the receptors, hindering their activity. This mechanism could act as a back-up system for T cell killing when antibody production might otherwise lead to protection of the target from attack by T cells through the blockage of the surface antigens (10). Kikuchi et al (7) from a study of 30 patients concluded that nonresponsiveness to M. leprae antigen of LL patients was generated by elimination of responding T cells and that T8 suppressor cells play little role in it.
The impaired CMI observed in human leprosy could be due to massive infection by M. leprae, although the possibility is not excluded that some constitutional difference might favor the initial development of the lepromatous rather than the tuberculoid form of the disease (9). No explanation for this difference of host behavior has been found as yet. Rook (11) suggests two possibilities. Firstly, there may be a delay before the onset of CMI, so that when it appears the bacilli are already established in vulnerable tissues which are subsequently damaged by inflammation. Secondly the CMI could be directed against antigenic components which are not released by live organisms, so that the immunological attack occurs in the wrong places, around dead or leaking bacilli.
The presence of circulating IC formed during the course of the disease with specific mycobacterial antigens and their evoked antibodies could similarly bring about a suppression of the ADCC function particularly in the LL patients. Thus the disease may be impossible to be contained by the immunological defense system of the body under these circumstances. There is a definite relation between the decreased ADCC and increased bacterial counts. The deficiency in NK activity seen in patients with ENL cannot be attributed to any single mechanism. There is a possibility that decreased ADCC and NK cell activity may be inhibited but it is to be noted that ADCC depression is present in all types of leprosy, whereas NK cell activity depression occurs not in stable but in IC-mediated ENL. However, decrease in ADCC is part of the general immune suppression seen after injection of IC, which in turn was responsible for the enhanced bacterial counts.