Evidence for Extensive Duplication and Subfunctionalization of FCRL6 in Armadillo (Dasypus novemcinctus).

The control of infections by the vertebrate adaptive immune system requires careful modulation to optimize defense and minimize harm to the host. The Fc receptor-like (FCRL) genes encode immunoregulatory molecules homologous to the receptors for the Fc portion of immunoglobulin (FCR). To date, nine different genes (FCRL1-6, FCRLA, FCRLB and FCRLS) have been identified in mammalian organisms. FCRL6 is located at a separate chromosomal position from the FCRL1-5 locus, has conserved synteny in mammals and is situated between the SLAMF8 and DUSP23 genes. Here, we show that this three gene block underwent repeated duplication in Dasypus novemcinctus (nine-banded armadillo) resulting in six FCRL6 copies, of which five appear functional. Among 21 mammalian genomes analyzed, this expansion was unique to D. novemcinctus. Ig-like domains that derive from the five clustered FCRL6 functional gene copies show high structural conservation and sequence identity. However, the presence of multiple non-synonymous amino acid changes that would diversify individual receptor function has led to the hypothesis that FCRL6 endured subfunctionalization during evolution in D. novemcinctus. Interestingly, D. novemcinctus is noteworthy for its natural resistance to the Mycobacterium leprae pathogen that causes leprosy. Because FCRL6 is chiefly expressed by cytotoxic T and NK cells, which are important in cellular defense responses against M. leprae, we speculate that FCRL6 subfunctionalization could be relevant for the adaptation of D. novemcinctus to leprosy. These findings highlight the species-specific diversification of FCRL family members and the genetic complexity underlying evolving multigene families critical for modulating adaptive immune protection.

Integrated pathways for neutrophil recruitment and inflammation in leprosy.

Neutrophil recruitment is pivotal to the host defense against microbial infection, but it also contributes to the immunopathology of disease. We investigated the mechanism of neutrophil recruitment in human infectious disease by means of bioinformatic pathways analysis of the gene expression profiles in the skin lesions of leprosy. In erythema nodosum leprosum (ENL), which occurs in patients with lepromatous leprosy and is characterized by neutrophil infiltration in lesions, the most overrepresented biological functional group was cell movement, including E-selectin, which was coordinately regulated with interleukin 1beta (IL-1beta). In vitro activation of Toll-like receptor 2 (TLR2), up-regulated in ENL lesions, triggered induction of IL-1beta, which together with interferon gamma induced E-selectin expression on and neutrophil adhesion to endothelial cells. Thalidomide, an effective treatment for ENL, inhibited this neutrophil recruitment pathway. The gene expression profile of ENL lesions comprised an integrated pathway of TLR2 and Fc receptor activation, neutrophil migration, and inflammation, providing insight into mechanisms of neutrophil recruitment in human infectious disease.

T lymphocyte subpopulations in leprosy patients and their relation with circulating immune complexes.

The possible relationship between circulating immune complexes (CIC) and peripheral T lymphocyte populations was studied in thirteen active multibacillary leprosy (10 lepromatous--LL--and 3 borderline lepromatous--BL--) and 19 matched controls. Theophylline-resistant T cells (The-R, a lymphocyte subpopulation displaying helper activity on B cells) and total T cells were assessed by means of the E rosette technique, with and without previous theophylline incubation, 1h 37 degrees C, respectively. CIC were quantified by 125I-C1q binding test. Although leprosy patients showed a statistical non significant light depression in total T cells the remarkable variability in circulating levels of The-R T cells enabled us to separate them into two well delineated groups (in relation to this variable p less than 0.001) with no difference in age, sex and bacteriologic state: a) leprosy patients with The-R T cells proportionally conserved (6LL and 2BL); b) leprosy patients with The-R T cells proportionally depressed (4LL and 1BL). Patients belonging to the latter group showed the highest statistically significant levels of CIC. Even though we do not discard an unknown factor being responsible for our findings, we believe that this inverse relationship between elevated CIC and depressed The-R circulating T cells might be representing a lower helper activity on antibody synthesis intending to reduce its excessive production.

In vitro and in vivo experiments with the new inhibitor of mycobacterium leprae brodimoprim alone and in combination with dapsone.

The antibacterial effect of brodimoprim alone and in combination with dapsone has been studied in vitro in cell-free systems and in whole mycobacterial cells as well as in vivo in mice and humans. The obtained inhibitory effects in vitro and in vivo against model mycobacterial strains and M. leprae, the pharmacokinetic properties in human and its synergistic effect with the most used drug in the chemotherapy of leprosy, dapsone, make brodimoprim a promising candidate in the therapy of leprosy.

Correlation between macrophage activation and bactericidal function and Mycobacterium leprae antigen presentation in macrophages of leprosy patients and normal individuals.

The killing of Mycobacterium leprae by resting and gamma interferon (IFN-gamma)-activated macrophages in normal subjects and leprosy patients was assessed. Resting macrophages from normal individuals demonstrated the ability to kill M. leprae. For macrophages from tuberculoid patients, killing of M. leprae was only achieved in the presence of IFN-gamma, suggesting that initial T-cell activation occurs prior to the killing of M. leprae. In contrast, though activation with IFN-gamma rendered the lepromatous macrophages microbicidal, it failed to induce lymphocyte proliferation, suggesting a defect at either the antigen-presenting cell or the lymphocyte level or both. The concept that T-cell anergy is primarily due to lack of lymphokine generation was ruled out by our results, since responsiveness was restored in only a small proportion of lepromatous patients after exogenous lymphokine addition. In conclusion, this study demonstrated that killing and antigen presentation are two independent events. It appears that the ability of the macrophages per se to kill M. leprae may be of greater importance than lymphocyte-mediated activation for protection against M. leprae infection.

Correlation of in vitro Fc receptor assay with in vivo mouse foot pad method.

Resistant strains of M. leprae have been reported to the various antileprosy drugs. There is currently no accepted test to identify the susceptibility pattern of M. leprae to the drugs in a short period. The only accepted test is the mouse foot method which takes a long period to yield results. The Fc receptor assay using the ability of viable M. leprae to alter the membrane of the macrophage is well established. It takes only ten days and is inexpensive. In 6 cases of leprosy patients the susceptibility pattern was worked out both with the in vitro Fc receptor assay and the vivo in mouse foot method The results correlated very well leading to the fact that the assay system is reliable. Hence it can be used not only to study the status of a patient, but also to shortlist the number of compounds to be tested on the mouse foot pad as anti-leprosy drug candidates.

Rheumatoid factors in subacute bacterial endocarditis and other infectious diseases.

Rheumatoid factors (RF) occur during the course of various infections such as leprosy, infective endocarditis, tuberculosis, trypanosomiasis, visceral larva migrans, infectious mononucleosis, influenza A, hepatitis A or cytomegalovirus. When first described it seemed logical to assume that host-self-immunization with autologous immune complexes provided the initial stimulus for RF production. Subsequently extensive characterization of bacterial, parasitic and viral Fc receptors has suggested an alternative explanation for rheumatoid factor associated with infections. It seems possible that patients make an initial immune response to infecting agent Fc receptors and that anti-anti-Fc receptors or anti-idiotypes either then directly stimulate rheumatoid factor production or are themselves rheumatoid factors. Such a hypothesis might also be applied to rheumatoid arthritis itself where either infecting agent or autologous cell Fc receptors could be the initial immunizing epitopes involved in rheumatoid factor production.

Lepromin-induced lymphoproliferative response of experimental leprosy monkeys: regulatory role of monocyte and lymphocyte subsets.

We investigated the immunological status of seven normal, control Mangabey monkeys and 23 Mangabey monkeys experimentally inoculated with Mangabey-origin Mycobacterium leprae. Clinically, these monkeys were divided into three broad groups: a recently inoculated group, a resistant group, and a susceptible group. The resistant group included 11 monkeys, seven of which showed no clinical sign of disease to date and four of which had shown local disease that partially regressed spontaneously. The susceptible group included eight monkeys, five of which have disseminated disease and three with local but stable disease. When peripheral blood mononuclear cells of these monkeys were cultured with Dharmendra-type human lepromin, one of seven normal monkeys, four of four of the recently inoculated group, seven of 10 resistant monkeys, and three of eight susceptible monkeys showed significant responses. In this experimental monkey model, we studied possible regulatory mechanisms by using OKT4- and OKT8-enriched lymphocytes, and Fc receptor-positive (FcR+) and FcR- monocyte (M phi) subsets. The OKT4+ subset was the main lepromin-responsive cell type. High percentages of OKT8+ cells showed a good negative correlation with the lymphoproliferative responses of T-enriched cells supplemented with unfractionated M phi. But the depletion of OKT8+ cells could not increase the response of nonresponding monkeys' lymphocytes. The resistant group and susceptible group did not differ in their percentages of OKT8+ cells. Because OKT8+ cells negatively regulate the response of lymphocytes and OKT4+ cells are the main responding cells, OKT8+ cells are phenotypically and functionally suppressor cells and OKT4+ cells are the helper/inducer cell population in this system. The FcR- M phi population mainly includes antigen-presenting activity, but high percentages of FcR- M phi showed a significant negative correlation with lymphoproliferative responses in the resistant group. A weak but significant lymphocyte response to Dharmendra lepromin was obtained by depleting FcR+ M phi from cultures of some susceptible monkeys, whereas lymphocytes of other susceptible monkeys remained unresponsive to lepromin. By these criteria, we could find an array of immunological defects in monkeys with experimental leprosy. The data suggest that some immunological defects may exist in the OKT4+ lymphocytes or FcR- M phi of leprosy monkeys.

Regulatory role of FcR+ and FcR- monocyte subsets in Mycobacterium leprae-induced lymphoproliferative response in vitro.

We investigated nine rhesus monkeys (Macaca mulatta) inoculated with Mycobacterium leprae and three normal human contacts. Peripheral blood monocytes were separated into Fc receptor positive (FcR+) and Fc receptor negative (FcR-) fractions, and their regulatory role in the lymphoproliferative response in vitro to M. leprae was studied. FcR- monocytes had strong antigen presentation activity and produced no suppressor effect while FcR+ monocytes had weak antigen presentation activity and produced a non-specific suppressor factor spontaneously. With this assay system we determined that M. leprae-inoculated rhesus monkeys could be divided into three groups: good responders, very weak responders, and non-responders.

M. leprae phagocytosis and its association with membrane changes in macrophages from leprosy patients.

Abnormal phagocytosis of Mycobacterium leprae by macrophages of lepromatous patients was demonstrated under various conditions. The largest proportion of macrophages with an excessive bacterial load belonged to the lepromatous group of patients. Lepromatous macrophages treated with Cytochalasin B, an inhibitor of phagocytosis, exhibited a significantly lower degree of ingestion of heat-killed organisms whereas uptake of 'viable' organisms was not affected to the same extent. Regulation of phagocytosis was studied by noting the rate of phagocytosis of M. leprae after the ingestion of a primary particle viz carbonyl iron. Solely in lepromatous macrophages, phagocytosis of carbonyl iron did not result in a decreased uptake of M. leprae implying aberrant phagocytic activity. Lastly, excessive phagocytosis was always noted in macrophages of familial contacts of leprosy patients who displayed decreased Fc receptor expression after M. leprae ingestion. This is of interest since phagocytosis, like Fc receptor expression, is a membrane dependent event and other membrane associated defects have been recognized by us earlier in lepromatous macrophages.

Mycobacterium leprae-induced alterations in macrophage Fc receptor expression and monocyte-lymphocyte interaction in familial contacts of leprosy patients.

Macrophage Fc receptor expression and monocyte-lymphocyte interaction in the presence of Mycobacterium leprae were examined in familial contacts of leprosy patients. Defective M phi functions similar to those of borderline and lepromatous patients could be observed in approximately 71% of consanguineous contacts and 43% of spouses of index patients. Although the values in the latter group were markedly lower than those of the consanguineous contacts, they tended to be higher than those of normal individuals (20%). These in vitro M phi functions were independent of age, sex, and age at onset of exposure and were only weakly associated with duration of exposure. The outcome of the monocyte-lymphocyte interaction test paralleled to a large extent the in vivo Mitsuda lepromin response. Four contacts with defective M phi functions also showed signs of leprosy. The value of these in vitro tests as markers of 'susceptibility' could therefore prove significant.

Mechanism of immunosuppression in leprosy--macrophage membrane alterations.

L-Lysate induced macrophage membrane alteration was studied using 3 membrane markers: (i) Fc receptor, (ii) Concanavalin A (Con A) receptor, and (iii) M. leprae adherence to macrophage membrane. The data indicate that L-lysate induces membrane perturbation of normal macrophages. The alteration can be reversed with trypsin and colchicine. Membrane alteration observed may lead to defective macrophage participation in a cell-mediated immune reaction.

In vitro studies on the effect of levamisole in lepromatous leprosy.

Our previous studies have demonstrated a defective macrophage response to M. leprae in lepromatous leprosy patients. In the present study we report the restoration of Fc receptor and HLA-DR antigen expression as well as antigen specific macrophage-lymphocyte interaction on treatment with levamisole in vitro. These results indicate that levamisole activates the macrophages which in turn results in an improved cell mediated immune response in lepromatous leprosy. This may also be applicable in other disease situations.

Alterations in the membrane of macrophages from leprosy patients.

Macrophage cultures pulsed with viable Mycobacterium leprae were assessed for erythrocyte rosetting in three groups of individuals, i.e., normal subjects, and tuberculoid and lepromatous patients. Of these, only the lepromatous group showed a reduction in rosetting ability after infection with M. leprae. The specificity of such a reduction pattern was confirmed by using various mycobacteria to infect the macrophages. A threshold effect was noted in all three groups. Although a reduction was obtained in the amount of rosetting of macrophages from lepromatous patients with 10(4) acid-fast bacilli per culture, tuberculoid and normal macrophages resisted such an effect with as large a dose as 20 X 10(6) to 30 X 10(6) and 30 X 10(6) bacilli per culture, respectively. The M. leprae-caused alterations in macrophages from lepromatous patients were reversible by treatment with trypsin and colchicine. Cytochalasin B and Tween 80 were unable to alter the pattern. Treatment of cells with neuraminidase was inconclusive since it enhanced rosetting values of both control and infected cultures. These manipulations were significant in elucidating the target point of the host (macrophage) and parasite (M. leprae) interaction and in delineation of the external and internal effects upon the macrophages. Both M. leprae and macrophages were participants in Fc reduction, as treatment of the former with rifampicin and of the latter with cyclocheximide significantly augmented the rosetting ability. In conclusion, it appears that M. leprae, upon entering a lepromatous macrophage, initiates the production of a protein which acts via the microtubules to alter membrane topography. It is possible that the altered membrane prevents effective macrophage-lymphocyte interaction. This could be one of the mechanisms by which cell-mediated immunity is suppressed in lepromatous leprosy.

Activated suppressor T cells in leprosy

Leprosy is a spectral disease in which the patients at the lepromatous end display selective immunologic unresponsiveness to antigens of M. leprae. We have previously explored the possibility that the anergy in lepromatous leprosy mediated by suppressor cells. An in vitro system was developed in which lepromin induces suppression of the mitogenic response of lepromatous and borderline but not tuberculoid leprosy patients to concavalin A.

The monocyte-macrophage system in granulomatous inflammation.

Granulomas may be immunologically induced or non-immunologically induced. In immunologically induced granulomas cells of the monocyte-macrophage series take on the appearance of epitheloid cells. Ultrastructurally epithelioid cells may have a secretory appearance with much rough endoplasmic reticulum or take on highly degenerate vesicular appearance. Other epithelioid cells look like activated macrophages. Secretory epithelioid cells may be found associated with acute local inflammation as in borderline tuberculoid leprosy in reaction, the lepromin reaction, following injection of BCG vaccine and in experimental zirconium granulomas. In these situations there may also be strong histological and biochemical evidence of increased fibroblast activity and collagen synthesis. It is suggested that these cells are actively secreting a fibroblast-activating factor. Epithelioid cells may lose their Fc receptors, undifferentiated macrophages in lepromatous leprosy can lose their C3 receptors. It is suggested that in a number of situations granuloma formation may be associated with complement activation through the alternative pathway as in the case of mycobacterial granulomas. Toxic granulomas produced by metals may be caused by C3 being split by plasmin after conversion from plasminogen by activation of the Hageman factor.

Peripheral blood monocyte function in leprosy patients.

The activities of monocytes from lepromatous (LL) and tuberculoid (TT) leprosy patients were studied in a variety of in vitro systems. In assays of receptor activity, increased densities of Fc and C3b receptor sites were observed in monocytes of LL and TT patients as compared to normals. No differences were observed between the polar forms of the disease. Similar results were obtained in the nitroblue tetrazolium (NBT) reduction test. Glucosamine incorporation by monocytes from LL and TT patients was not significantly different from that by monocytes from normal controls. A diminished monocyte spontaneous migration and chemotactic activity to lymphocyte derived chemotactic factor (LDCF) was found in both forms of the disease. Plasma inhibitory factor of monocyte chemotaxis was more evident in the lepromatous form of the disease.

Biochemical alteration in cells following phagocytosis of M. leprae--the consequence--a basic concept.

When macrophages from lepromatous leprosy patients are exposed to M. leprae, the macrophages show reduced protein synthesis. Such a phenomenon is not seen with macrophages from tuberculoid patients or normal individuals. M. leprae phagocytized by Schwann cells affect the incorporation of DNA precursor in the cells, leading to failure of Schwann cell association with axons in in vitro cultures. These 2 observations form a basis of proposing that basic biochemical events take place when M. leprae are associated with host cells, which in turn can be amplified to physiologically functional defects.