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Antigen-Coated Latex Particles As a Model System for Probing Monocyte Responses in Leprosy RUMINA S

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Antigen-Coated Latex Particles As a Model System for Probing Monocyte Responses in Leprosy RUMINA S 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 Leprosy 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 Mycobacterium 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 lepromatous leprosy patients than in cells from tuberculoid leprosy 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 Mycobacterium bovis 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- infection with Mycobacterium tuberculosis (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 bacteria 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 armadillo-derived material may have variable contamination armadillo-derived M. leprae, at concentrations of 50 to 200 with foreign proteins. 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.
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