INFECrION AND IMMUNITY, Sept. 1993, p. 3724-3729 Vol. 61, No. 9 0019-9567/93/093724-06$02.00/0 Copyright C 1993, American Society for Microbiology Antigen-Coated Latex Particles as a Model System for Probing Monocyte Responses in RUMINA S. HASAN,1* HAZEL M. DOCKRELL,2 SARWAT JAMIL,1 THOMAS J. CHIANG,3 AND RABIA HUSSAIN1 Department ofMicrobiology, Aga Khan University, Stadium Road,1 and Marie Adelaide Leprosy Centre, Mariam Manzil,3 Karachi 5, Pakistan, and Department of Clinical Sciences, London School ofHygiene and Tropical Medicine, London WCJE 7HT, United Kingdom2 Received 2 November 1992/Returned for modification 2 February 1993/Accepted 21 June 1993 To study responses to leprae antigens, we developed an in vitro model system in which latex particles coated with M. keprae sonic extract (MLSON) antigen were presented to monocytes. Uptake and oxidative response as measured by superoxide production to these antigens were investigated. Phagocytosis of MLSON-coated particles was greater than that of control particles in monocytes from both leprosy patients and controls from leprosy-endemic areas; uptake of MLSON-coated particles was higher in monocytes from patients than in cells from patients and controls. In both patients and controls, uptake of latex particles coated with leprosy antigens triggered very little reduction of nitroblue tetrazolium although the cells were capable of mounting a respiratory burst. Antigen-coated latex particles can therefore be used as a tool to investigate monocyte responses to M. leprae and individual recombinant antigens.

The monocyte/macrophage is a central cell in the immune MATERIALS AND METHODS response to mycobacteria. In vitro macrophages have been shown to inhibit the growth of BCG Patients and controls. Untreated leprosy patients, present- (27) and Mycobacterium microti (30). On the other hand, ing at the Marie Adelaide Leprosy Centre, Karachi, Paki- macrophages have been implicated in the establishment of stan, and normal controls from leprosy-endemic areas, em- with Mycobacterium (6). In leproma- ployees of the Aga Khan University, Karachi, Pakistan, tous leprosy, the macrophages contain higher numbers of were used. The leprosy patients were diagnosed clinically by than are found in cells from tuberculoid leprosy bacterial counts on slit skin smears and histologically by patients, possibly because of increased uptake of bacteria, using a 4-mm punch skin biopsy taken from the edge of an decreased killing, or both. Using live Mycobacterium leprae, active lesion, according to the Ridley-Jopling classification Mistry et al. (17) showed that monocytes from lepromatous (22). The majority of both patients and controls have not patients acquired a greater bacterial load than do those from received BCG vaccination, but tuberculosis is highly en- tuberculoid patients or normal controls. Monocytes or mac- demic in Karachi. rophages from lepromatous leprosy patients are able to kill Antigen coating of latex particles. A 5% suspension of other bacteria such as Listeria monocytogenes, Staphylo- carboxylated latex particles of 1.1-p,m diameter (Seragen, coccus aureus, or Proteus vulgaris (5) and can be activated Indianapolis, Ind.) was used. Aliquots (200 ,ul) of latex by gamma interferon to kill legionellae (13). There are two particles were washed three times with 0.1 M carbonate reports that blood-derived macrophages from lepromatous buffer (pH 9.6) and then three times with 0.02 M phosphate leprosy patients were unable to kill M. leprae, unlike those buffer (pH 4.5) by adding buffer to the particles and spinning from tuberculoid patients or healthy controls, as assessed by the mixtures at 12,000 rpm. The particles were then resus- lysis (1) or by a reduction in uptake of tritiated thymidine pended in 500 ,ul of phosphate buffer, and 500 ,ul of 2% (21); other studies have failed to confirm these findings (4, 9). carbodiimide [1-(3-dimethylaminopropyl)-3-ethyl carbodiim- There are a number of technical difficulties encountered ide hydrochloride; Sigma Chemical Co., St. Louis, Mo.] was when whole M. leprae is used in such studies. First, the added drop by drop. The tubes were mixed at room temper- small size of the bacteria and their tendency to clump makes ature for 3 to 4 h. Unreacted carbodiimide was removed by counting by conventional microscopy difficult. Second, bac- washing the particles three times in 0.02 M phosphate buffer teria derived from lepromas contain a mixture of live and (pH 7.4). The antigen, M. leprae sonic extract (MLSON; dead organisms, which may have different properties, while batch CD 75), a soluble sonic extract of cobalt-irradiated -derived material may have variable contamination armadillo-derived M. leprae, at concentrations of 50 to 200 with foreign . In addition, the response to individual ,ug/ml in a final volume of 1 ml was added, and the mixtures components of M. leprae needs to be analyzed. We have were incubated with mixing overnight at room temperature. now developed a system in which mycobacterial antigens are Optimal coating was achieved with 200 ,ug of MLSON per coupled to latex particles and have used it to assess phago- ml. To block unreacted sites, 20 ,ll of ethanolamine (2- cytosis and oxidative response to M. leprae antigens by aminoethanol; Sigma) was added, and the particles were monocytes in leprosy. mixed for 1 h. After three washes in phosphate buffer (pH 7.4), any remaining sites were blocked by incubating the particles in 1 ml of 0.25% bovine serum albumin (BSA) in phosphate buffer. After a final three washes, the particles were stored as a 1% solution at 4°C in 0.02 M phosphate * Corresponding author. buffer (pH 7.4) with 8 mg of NaCl per ml, 10 mg of BSA per 3724 VOL. 61, 1993 SYSTEM FOR PROBING MONOCYTE RESPONSES IN LEPROSY 3725 ml, and 5% glycerol. Beads were also coated with BSA [NBT] when stimulated with phorbol myristate acetate (Sigma). Control particles were incubated in buffer alone. [PMA]). Assessment of antigen coating of latex particles. (i) Enzyme- Phagocytosis assay. A suspension of latex particles (3 x 107 linked assay. The antigen-coated latex particles were washed to 4 x 107 per well) in RPMI containing 10% heat-inactivated five times in 0.02 M phosphate-buffered saline (PBS) (pH fetal calf serum (FCS; Flow Laboratories) was added to each 7.4) containing 0.05% Tween 20 (Sigma) (PBS-Tween). The monocyte monolayer and incubated for 2 h at 37°C. Non- particles were then diluted in PBS containing 1% BSA and phagocytosed beads were washed off with warm RPMI, and 0.05% Tween 20 (antibody diluent) and incubated in a the cells were fixed with methanol and stained with 10% high-titered pool of leprosy sera known to contain immuno- Giemsa solution. Phagocytosis was assessed in terms of the globulin G (IgG) anti-MLSON antibodies (12) diluted 1/100 percentage of phagocytic cells or in terms of the number of for 1 h at 37°C and overnight at 4°C. They were washed five particles ingested per phagocytic cell (1 to 5, 6 to 15, or >15 times in PBS-Tween, blocked with PBS containing 5% BSA particles per cell). MLSON-coated latex particles were for 1 h at 37°C, and washed again five times. The particles opsonized by incubation in a pool of high-titer antibody- were then incubated for 2 h at 37°C with alkaline phos- positive lepromatous leprosy serum diluted 1/100 for 2 h at phatase-labelled goat anti-human IgG (Sigma). After a fur- 37°C and then washed in Hanks balanced salt solution ther five washes, color was developed by adding phos- (HBSS). In some experiments, whole armadillo-derived co- phatase substrate (Sigma), and the reaction was stopped by balt-irradiated M. leprae was used at a concentration of 8 x adding 100 ,ul of 3 M NaOH. The tubes were centrifuged, and 108 per ml, the incubation time was increased to 4 h, and the supernatants were read in an enzyme-linked immunosor- uptake was assessed microscopically after staining with bent assay (ELISA) reader at 410 nm. modified Ziehl-Neelsen stain. (ii) Agglutination assay. A 0.1% suspension of antigen- Superoxide anion production. The production of superox- coated latex particles was washed five times in PBS-Tween. ide anions was measured by the reduction of NBT (Sigma). After overnight incubation, the adherent monocyte mono- The agglutination assay was performed in round-bottom layers were washed with warm HBSS without phenol red microtiter plates containing 25 pl of antigen-coated latex, 25 (Flow Laboratories). A suspension of 1.2 x 108 to 1.6 x 108 pl of 0.2 M 2-amino-2-methyl-1-propanol-HCl (Eiken Chem- latex particles in phenol-red free HBSS containing 10% FCS ical Co., Tokyo, Japan), and 25 ,ul of a serum known to and 1 mg of NBT per ml was added to the cells (250 ,ul per contain a high titer of IgM antibodies to MLSON. A normal well) and incubated for 1 h at 37°C. PMA (Sigma) at a final serum pool was used as a negative control. The test was read concentration of 1 Fg/ml was used as a positive control. after overnight incubation at room temperature. The agglu- After incubation, the cells were washed with HBSS and tination index was read according to standard criteria for counterstained with 2% methyl green for 40 min. A minimum agglutination, in which the extent of mat or button formation of 200 phagocytic cells were counted on each coverslip, and was assigned a value of 3 (total formation of mat), 2 (75 to the percentage of cells reducing NBT was expressed as a 100% mat formation), 1 (50 to 75% mat formation), 0.5 (25 to fraction of the total phagocytic cells. For PMA, the percent- 50% mat formation), or 0 (button formation). An agglutina- age of cells reducing NBT was expressed as a percentage of tion index of 1 or greater is considered positive. The agglu- the total adherent cells. tination assay was performed on each batch of antigen- coated particles. To assess specificity of the agglutination assay, agglutination with five IgM antibody-positive sera RESULTS from BL or LL leprosy patients in the presence and absence Detection of antigen on latex particles. To assess antigen of soluble antigen was tested, using previously optimized coating, an indirect ELISA method using a leprosy serum concentrations of MLSON and an irrelevant Toxoplasma pool with high titers of IgG antibodies to MLSON was used, antigen supplied by M. Lunde, National Institutes of Health, with a normal serum pool as a control. Antigen-coated latex Bethesda, Md. bound antibodies in a highly specific manner; in addition, Monocyte culture. Heparinized venous blood (20 IU of there was no nonspecific binding of the antibodies to the heparin per ml; Evans Medical Ltd., Horsham, United BSA-coated or control latex particles (Fig. 1). Increasing Kingdom) was diluted 1:1 with warmed RPMI 1640 tissue binding was detected at coating antigen concentrations of up culture medium containing 10 mM N-2-hydroxyethylpipera- to 200 ,g/ml without a concomitant increase in nonspecific zine-N'-2-ethanesulfonic acid (HEPES), 0.2 mM L-glu- binding; therefore, this concentration was used in all subse- tamine, 25 U of penicillin per ml, and 25 pg of streptomycin quent experiments. The antigen coating assessed by an per ml and separated on Histopaque 1077 (Sigma). After agglutination assay was highly reproducible in eight sequen- three washes, 106 peripheral blood mononuclear cells in tial batches, all serum titers being within 1 standard devia- RPMI containing 20% autologous plasma (heat inactivated at tion of the mean (results not shown). 56°C for 30 min) were added to 13-mm-diameter coated Lux Another stringent measure of specificity of antibody bind- plastic coverslips (Flow Laboratories, Hertfordshire, United ing to antigen bound on a solid matrix is competitive Kingdom), and monocytes were allowed to adhere for 1 h at inhibition with soluble antigen. Figure 2 shows inhibition of 37°C in an atmosphere of 5% CO2 in air. After the nonad- the agglutination reaction by the specific antigen, MLSON, herent cells were washed off with warm RPMI, fresh tissue but not by an irrelevant soluble antigen preparation from culture medium was added and the cells were incubated Toxoplasma gondii with five separate lepromatous leprosy overnight. In some experiments, 20% heat-inactivated hu- sera. These standardized latex particles were then used to man AB serum (Flow Laboratories) was used instead of probe monocyte function in leprosy patients. autologous plasma. Before use, the adherent cells were again Phagocytosis of antigen-coated latex by monocytes from washed with warmed medium. The purity of the monocyte leprosy patients and controls. Monocyte monolayers from population was assessed by staining for nonspecific esterase both lepromatous and tuberculoid leprosy patients and from (14); 80 to 90% of the adherent cells were positive (although normal controls were tested for the ability to phagocytose nearly 100% of the cells reduced nitroblue tetrazolium MLSON-coated particles. In all three groups, the MLSON- 3726 HASAN ET AL. INFECT. IMMUN.

TABLE 1. Comparative uptake of M. leprae antigens in leprosy patients and in controls 2.00 - Patients Normal Untreated Untreated controls #*% 1.50- Prepn lepromatous tuberculoid a 0a Uptakea Uptake Uptake n n±+ SD n +SD n + SD ; 1.00 MLSON-coated 9 34.0'c + 14.5 10 20.3"b 9.0 11 20.1" ± 8.0 a latex latex 9 20.1 + 10.5 10 14.7 ± 7.6 11 12.3 ± 5.5 0.50 - Control M. leprae 4 39.4 ± 2.9 5 33.4 + 5.4 4 28.0 ± 2.0 a Percent phagocytic celis expressed as group mean. b Significantly greater than phagocytosis of control latex (P < 0.02; 0.00 - Wilcoxon signed rank test). 0.0 0.1 0.2 c Significantly greater than uptake of MLSON latex by untreated leprosy patients or normal controls (P < 0.01; Mann-Whitney test). LATEX CONC. (S) FIG. 1. ELISA to demonstrate coating of MLSON on latex particles. MLSON-coated particles (closed circles) were compared focus, indicating that the beads were phagocytosed rather with BSA-coated (open circles) and control (dotted line) latex than attached, although this has not been formally proved. particles in terms of reactivity with an antibody-positive serum pool Comparison of uptake between patients and controls from diluted 1/30. Reactivity was compared in an enzyme-linked assay endemic areas showed higher phagocytosis of MLSON- using three different particle concentrations, 0.01, 0.05, and 0.25%. Results are expressed as optical densities (OD) at 410 nm. Assays coated particles by monocytes from lepromatous patients were carried out in triplicate; error bars indicate standard errors compared with monocytes from tuberculoid patients or from around the means. controls. Interestingly, a similar trend was observed with the uncoated control particles and with whole M. leprae (Table 1). Some of this uptake may have been due to some opsonization of the particles by cross-reacting antibodies in coated particles were phagocytosed to a greater extent than the FCS. However, since the conditions used were the same the control particles were (Table 1). The increase was seen for all three study groups, this could not explain the greater not only in the total percentage of phagocytic cells but also uptake in lepromatous patients. Moreover, when MLSON- in the number of particles phagocytosed per cell. In the coated particles opsonized with lepromatous serum were lepromatous group, 9.41% ± 9.23% of the cells phagocy- used, phagocytosis was increased and was still significantly tosed more than five MLSON-coated particles, while only higher in the lepromatous patients (61.2% ± 14.0% of 2.93% ± 4.08% of the cells phagocytosed more than five phagocytic cells) than in the controls (46.2% ± 15.0% of control particles (P = 0.004; Wilcoxon signed rank test). To phagocytic cells) (P > 0.01; Mann-Whitney test). Thus, any ensure that the particles counted were actually phagocy- opsonization by the FCS was minimal, and there must be tosed and not merely attached to the membrane, the assays additional functional differences between the monocytes in were carried out at 4 and 37°C in three controls. In all cases, lepromatous patients and controls. minimal binding of particles to the monocytes was observed Superoxide anion production by monocytes in response to at 4°C; at 37°C, particles could be seen in different planes of phagocytosis. Monocytes from leprosy patients and controls were able to trigger a respiratory burst in response to PMA, with a mean response rate of 95% of the cells reducing NBT in all groups. The system was also able to detect NBT reduction around BSA-coated latex. In comparison with PMA- and BSA-coated particles, MLSON-coated latex re- sulted in a lower rate of NBT reduction (Fig. 3). The spontaneous reduction of NBT by monocytes after overnight incubation was extremely low, with <5% of the monocytes being positive in a 1-h incubation period. The low respiratory burst response to MLSON-coated latex was also seen in the larger donor panel; in most donors, the response to MLSON-coated particles was not higher than the response to control particles, and with the excep- tion of two normal controls, the percentage of cells reducing NBT was below 10% in all donors (Fig. 4). Thus, the LS2 LS7 LS26 LS47 LS8O MLSON-coated particles appeared to be phagocytosed via a Antibody positive sera receptor whose modulation does not induce a respiratory burst. FIG. 2. Specificity of agglutination of antigen-coated latex by soluble antigen. Insolubilized MLSON antigen on latex particles was reacted with antibodies in the presence of 20 p.g of specific DISCUSSION soluble antigen per ml, MLSON (open bars), an irrelevant antigen from T. gondii (hatched bars), or buffer (solid bars). The agglutina- For studies of host interactions with M. leprae, the model tion index against each of five antibody-positive sera obtained in a closest to the in vivo situation is one using whole bacteria. representative experiment is shown. Since M. leprae cannot be grown in culture, such studies use VOL. 61, 1993 SYSTEM FOR PROBING MONOCYTE RESPONSES IN LEPROSY 3727

100 50 Lepromatous patients I-- P. 0 U 40 = * 60 - a 30 03U 400 lo 0 0 L 20 20 - c; A. pt 10 0 - .2 1 2 3 0 Donors 511 512 515 517 526 528 529 535 549 FIG. 3. Oxidative responses to phagocytosed latex particles and PMA. The oxidative responses to MLSON-coated (open bars) and Donors BSA-coated (hatched bars) latex particles and to PMA (solid bars) were compared in monocytes from three controls. The results, expressed as the percentage of phagocytic cells reducing NBT or of 50 total monocytes reducing NBT for the PMA responses, represent the means and standard errors of triplicate wells. Tuberculoid patients I 40 organisms obtained from infected armadillo tissue (liver or r 30 spleen) or extracted from human lepromas. The different es sources and methods of isolation, however, are a source of ' 20 variation; as many of the M. leprae antigens recognized to date are heat shock proteins produced by the organism in IL I .r response to stress (33, 34), one might expect variation in ." &ww their expression in different tissues. Differences in viability p10 of live organisms would also be expected. All of these 0 factors make it difficult to relate results from different studies using whole organisms. Furthermore, whole bacteria do not 508 509 510 513 514 516 519 520 521 522 allow analysis of the host interaction at the level of individ- ual components, which is important in order to identify Donors antigens important for virulence. To study the interaction of leprosy antigens with mono- cytes and macrophages, it is necessary to have a system in 50 which functional capabilities, including activation and sub- Normal controls sequent cellular events, can be quantitated. Some authors have used electron microscopy to assess phagocytosis ofM. 40 leprae (25) or labeled similar organisms with fluorescent dye to make quantitation by light microscopy easier (8). Antigen- g 30 coated latex particles have been used in other systems (10, 19) and have a number of advantages over the use of whole 020 bacteria. The particle size chosen here, 1.1-p.m diameter, U makes the refractile round particles easier to quantitate by IL l light microscopy than the slender M. leprae (3.0 by 0.3 ,um), which have a tendency to clump and therefore cannot be counted accurately. Induction of a respiratory burst in 10 response to phagocytosed particles can also be easily ob- served. The covalent linking of antigens to latex particles by N2 N3 N4 N6 N7 N8 N9 the carbodiimide method proved stable, and recognition of the antigens by antibodies in two assay systems proved that NI NS NION1I the proteins were present on the particles in an antigenic form. Finally, the system can be adapted to investigate the FIG. 4. Oxidative responses to phagocytosis of MLSON-coated responses to individual antigens, such as the M. (solid bars) and control (hatched bars) latex in monocytes from leprosy patients and controls. Results are expressed as the percent- leprae recombinant 18-kDa antigen (28), which was success- NBT. Means and standard same not age of phagocytic cells (PC) reducing fully coupled to latex by the technique (results errors of triplicate wells are shown for each donor. The mean shown). response rate of monocytes in all groups when tested with PMA as Phagocytosis of antigen-coated beads was greater in lep- a positive control was 95%. romatous leprosy patients than in those with tuberculoid disease or normal controls. Lepromatous leprosy patients 3728 HASAN ET AL. INFECT. IMMUN. have been shown to have 10- to 100-fold-higher concentra- We have described a system in which the macrophage tions of antibodies than tuberculoid patients do (11, 16). response to various antigen presented on latex beads can be Although the assays were performed in tissue culture me- analyzed. Phagocytosis of the particles may allow antigen dium containing FCS, passive absorption of antibodies to the processing and presentation in a manner more similar to monocyte Fc receptors may have occurred during the over- when whole bacteria are used than when soluble antigens are night incubation in medium containing autologous plasma. used. Other applications of the antigen-coated latex particles The absorbed antibodies would increase binding and phago- include analysis of cytokine release in response to the cytosis of antigen-coated particles. It is also possible that the particles. We are also using this system to compare the effect number of Fc receptors is increased on monocytes in lepro- of opsonization with antibodies from tuberculoid or lepro- matous leprosy patients, although viable M. leprae has been matous leprosy patients on phagocytosis and the respiratory reported to downregulate Fc receptors in lepromatous lep- burst. rosy (17). A number of studies have also noted increased uptake of M. leprae by cells from lepromatous patients (15, ACKNOWLEDGMENTS 17), although comparable uptake of This investigation received financial support from the UNDP/ pseudotropicalis by monocytes from leprosy patients and World Bank/WHO Special Programme for Research and Training in from controls has been observed (7). Tropical Diseases, from a WHO directors initiative grant, and from Enhancement of phagocytosis by antibody, however, can- an Aga Khan University seed money grant. not explain increased uptake of MLSON-coated particles We thank F. Firdausi, Q. Ehsan, Z. Oquali, M. Ali Khan, and S. compared with uncoated control particles by monocytes Mousa for help in collecting the clinical material used in the study, from tuberculoid leprosy patients and controls. This en- S. Lucas for performing the histological diagnosis, R. J. W. Rees for hancement may be due to interactions between components providing the MLSON, and J. Blackwell and J. Ellner for helpful of MLSON and receptors or molecules on the monocytes. discussions. The monocyte surface membrane contains a number of other REFERENCES receptors through which ligand binding and phagocytosis 1. Barbieri, T. A., and W. M. Correa. 1967. Human macrophage can occur. For example, Pneumocystis carinii is phagocy- culture. The leprosy prognostic test (LPT). Int. J. Lepr. 35:377- tosed via the macrophage (8), Leishmania 381. parasites are phagocytosed by gp63 binding to the CR3 2. Bullock, W. E., and S. D. Wright. 1986. 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K. ment receptors, as it has been shown that binding to this Armstrong, A. Warner, F. F. Richards, and R. M. Rose. 1991. receptor does not initiate a respiratory burst, and would Uptake of Pneumocystis carinii mediated by the macrophage argue against opsonization of the particles by cross-reactive mannose receptor. Nature (London) 351:155-158. antibodies in the FCS, leading to uptake via Fc receptors 9. Godal, T., and R. J. W. Rees. 1970. Fate of Mycobacterium (31, 32). The response to PMA was normal, as shown earlier leprae in macrophages of patients with lepromatous or tubercu- by Sharp and Bannerjee (26). In fact, the BSA-coated latex loid leprosy. Int. J. Lepr. 38:439-442. particles triggered more NBT reduction than did the parti- 10. Gudewicz, P. W., J. Molnar, M. Z. Lai, D. W. Beezhold, G. E. Siefring, R. B. Credo, Jr., and L. Lorand. 1980. 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