Publications

1996

1995

Gaynor, C D, F X McCormack, D R Voelker, S E McGowan, and L S Schlesinger. (1995) 1995. “Pulmonary Surfactant Protein A Mediates Enhanced Phagocytosis of Mycobacterium Tuberculosis by a Direct Interaction With Human Macrophages.”. Journal of Immunology (Baltimore, Md. : 1950) 155 (11): 5343-51.

During initial infection with Mycobacterium tuberculosis, bacteria that reach the distal airspaces of the lung are phagocytosed by alveolar macrophages in the presence of pulmonary surfactant. Here we have examined the role of surfactant-associated protein A (SP-A) in phagocytosis of the virulent Erdman strain of M. tuberculosis by human monocyte-derived macrophages (MDMs) and human alveolar macrophages (HAMs). Macrophage monolayers incubated with soluble SP-A from alveolar proteinosis patients (APP SP-A4) and recombinant rat SP-A (SP-Ahyp) demonstrated enhanced adherence of M. tuberculosis, 82 +/- 17% and 49 +/- 18%, respectively. Removal of SP-A from monolayers by washing before adding bacteria did not diminish the enhanced adherence. Fluorescence microscopy demonstrated that washed monolayers contained intracellular rather than surface-bound SP-A. These studies indicated a direct interaction between SP-A and the macrophage in mediating enhanced adherence of M. tuberculosis. Consistent with this interpretation, macrophage monolayers formed on human or rat SP-A (substrate SP-A) demonstrated enhanced adherence of M. tuberculosis to their apical surface (APP SP-A and native rat SP-A increased M. tuberculosis adherence by 102 +/- 16% and 102 +/- 25%, respectively). Electron microscopy demonstrated increased numbers of phagocytosed bacteria in APP SP-A-treated MDM cross-sections. SP-A proteins devoid of carbohydrate failed to enhance M. tuberculosis adherence to macrophages. In contrast, heat-denatured APP SP-A enhanced adherence of bacteria equivalent to that of intact glycoprotein. Thus, the carbohydrate moieties of SP-A appear to be critical in the SP-A-macrophage interaction. Finally, mannan and anti-mannose receptor Ab completely inhibited the enhanced phagocytosis of M. tuberculosis observed with APP SP-A, providing evidence for up-regulation of macrophage mannose receptor activity. These studies implicate SP-A as an important modulator of alveolar macrophage function that results in an enhanced potential for M. tuberculosis to gain access to its intracellular niche.

1994

We have previously determined that complement receptors on human mononuclear phagocytes and complement component C3 in nonimmune serum mediate phagocytosis of the intracellular bacterial pathogen Mycobacterium leprae, the agent of leprosy. We have also determined that C3 fixes selectively to the major surface glycolipid of M. leprae, phenolic glycolipid 1 (PGL-1). In this study, we have explored the role of natural antibody in nonimmune serum in C3 fixation and C1q binding to M. leprae and PGL-1. At serum concentrations within the range at which phagocytosis of M. leprae is maximal, C3 fixation was mediated by both the classical and the alternative complement pathways. At the low end of this serum concentration range (2.5%), C3 fixation was mediated predominantly by the classical pathway. Consistent with a role for both pathways, C3 fixation to M. leprae was enhanced by the addition of either pure C1q to C1q-depleted serum or pure factor B to factor B-depleted serum. C3 fixation to M. leprae was strictly antibody dependent regardless of the serum concentration used. C3 fixation to M. leprae occurred in nonimmune serum but not in agammaglobulinemic serum unless heat-inactivated nonimmune serum or small amounts of pure immunoglobulin G (IgG) or IgM were added. C3 fixation by both the alternative and the classical complement pathways was mediated by antibody, and the antigen-binding portion of the antibody molecule was required. C3, IgG, IgM, and C1q were readily detected on the surface of M. leprae. Consistent with the previously demonstrated exclusive role of the classical complement pathway in C3 fixation to PGL-1, C1q bound to PGL-1 in a dose-dependent fashion; C1q binding was evident in > 1.25% nonimmune serum. C1q binding to PGL-1 was strictly antibody dependent. When PGL-1 was incubated with pure C1q, little or no C1q bound to PGL-1 unless heat-inactivated nonimmune serum or pure IgG or IgM was added. When PGL-1 was incubated in nonimmune serum, C3 bound directly to PGL-1 and not to anti-PGL-1 antibody, since the amount of C3 bound to PGL-1 was not reduced by acid elution of the antibody. However, the amount of C3 bound to PGL-1 was markedly reduced by hydroxylamine treatment, providing evidence for C3 fixation via a covalent ester bond. Nonimmune serum contained antibody to all four major M. leprae surface carbohydrates. Relative to PGL-1, nonimmune serum contained more antibody to the other surface carbohydrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Schlesinger, L S, S R Hull, and T M Kaufman. (1994) 1994. “Binding of the Terminal Mannosyl Units of Lipoarabinomannan from a Virulent Strain of Mycobacterium Tuberculosis to Human Macrophages.”. Journal of Immunology (Baltimore, Md. : 1950) 152 (8): 4070-9.

Recent studies from this laboratory have demonstrated that macrophage phagocytosis of virulent strains (Erdman and H37Rv), but not the attenuated H37Ra strain of Mycobacterium tuberculosis, is mediated by phagocyte mannose receptors (MR) in addition to complement receptors (CR1 and the leukocyte integrins CR3 and CR4). Lipoarabinomannan (LAM) is a major surface lipoglycan of M. tuberculosis. LAM from the Erdman strain (Man-LAM) contains mannose oligosaccharides at the terminal portions of the molecule. This study investigated the ability of ManLAM to serve as a microbial ligand in adherence to human monocyte-derived macrophages (MDM). Polystyrene microspheres were coated with known amounts of purified ManLAM, LAM without the terminal mannosyl units from an avirulent mycobacterium (AraLAM), lipomannan (LM), or buffer and incubated with MDM monolayers in the absence of serum. The presence of LAM on microspheres was confirmed by indirect immunofluorescence studies. Microspheres coated with ManLAM demonstrated a more than threefold increase in adherence to MDM when compared with microspheres coated with AraLAM, LM, or buffer and the low levels of adherence of microspheres in the latter three groups were comparable. Compared with control monolayers, selective down-modulation of MDM MR on a mannan substrate abrogated the enhanced adherence of microspheres mediated by ManLAM. Adherence of microspheres coated with AraLAM, LM, or buffer was not influenced by MR modulation. To confirm the importance of the terminal mannosyl units of ManLAM in the enhanced adherence of ManLAM microspheres to MDM, these units were selectively removed by exomannosidase treatment. The structure of LAM products before and after enzyme treatment was confirmed by high performance anion exchange chromatography with pulsed amperometric detection. Removal of the terminal mannosyl units abolished the capacity of ManLAM to mediate enhanced adherence of microspheres to MDM. Finally, preincubation of Erdman M. tuberculosis with CS-40, a mAb directed against LAM, resulted in a consistent inhibition of adherence of the bacteria to MDM (up to 49% inhibition), confirming a role for ManLAM on intact bacteria in adherence to MDM. Thus, we provide evidence for a novel receptor-ligand pathway in phagocytosis of M. tuberculosis that consists of MR on macrophages and mannosyl units at the terminal end of ManLAM, a major microbial surface lipoglycan.

Gaynor, C D, R A Clark, F P Koontz, S Emler, B Hirschel, and L S Schlesinger. (1994) 1994. “Disseminated Mycobacterium Genavense Infection in Two Patients With AIDS.”. Clinical Infectious Diseases : An Official Publication of the Infectious Diseases Society of America 18 (3): 455-7.

Mycobacterium genavense is a recently defined fastidious organism that has been identified as a cause of disseminated infection in patients with AIDS. We report the cases of two patients who had advanced AIDS and a clinical syndrome of fever, anorexia, abdominal pain, diarrhea, and weight loss. In addition, splenomegaly and lymphadenopathy were prominent in both cases, and in one patient's case radiographic findings were suggestive of splenic abscesses. Mycobacteria isolated from specimens of blood and bone marrow grew in liquid media but not on solid media. The results of DNA probe tests for Mycobacterium tuberculosis and Mycobacterium avium complex were false-positive for both patients. After treatment of the broth cultures to lyse red blood cells, the results of DNA probe tests were negative for these pathogens. Amplification and sequencing of 16S rRNA with use of the polymerase chain reaction indicated that the mycobacterial isolates from both patients had sequences identical to those previously reported for M. genavense. One patient survived 5 months after diagnosis, the other 2 months after diagnosis; only one patient responded (transiently) to antimycobacterial chemotherapy.

1993

We have examined macrophage receptors that mediate phagocytosis of virulent strains (Erdman and H37Rv) and an attenuated strain (H37Ra) of the intracellular pathogen, Mycobacterium tuberculosis. Adherence of the three strains to monocyte-derived macrophages (MDM) is markedly enhanced (>threefold) in the presence of low levels of fresh serum and requires heat-labile serum components because heat inactivation of serum reduces adherence by 65 +/- 5 to 71 +/- 2%. In the presence and absence of serum, adherence of the three strains to MDM is comparable. By electron microscopy, all bacteria are ingested and reside in phagosomes. C receptors (CR) play an important role in adherence of the three strains to MDM in the presence and absence of serum. mAb against CR1, CR3, and CR4 inhibit adherence of Erdman M. tuberculosis in fresh serum by 75 +/- 3% and inhibit the low level of adherence of Erdman (71 +/- 13%), H37Rv (72 +/- 1%), and H37Ra (64 +/- 14%) M. tuberculosis in the absence of serum. Mannose receptors (MR) play an important role in mediating macrophage adherence of the virulent strains but not the attenuated strain of M. tuberculosis. Preincubation of MDM with soluble mannan or mannose-BSA consistently and significantly inhibits adherence of Erdman and H37Rv (up to 60 +/- 7%) but not H37Ra (0 +/- 1 to 5 +/- 5% enhancement of adherence) in the absence of serum. Down-modulation of macrophage MR on mannan substrates inhibits adherence of Erdman (52 +/- 8%) and H37Rv (55 +/- 6%) but not H37Ra (2 +/- 2% enhancement of adherence). Preincubation of MDM with soluble N-acetylglucosamine-BSA also significantly inhibits adherence of the virulent strains (42 +/- 3%). Preincubation of MDM with glucose-BSA minimally inhibits adherence of the three strains (2 +/- 4 to 12 +/- 5%). Anti-MR antibody inhibits adherence of Erdman (57 +/- 2%) and H37Rv (44 +/- 4%) but not H37Ra (4 +/- 5% enhancement of adherence). Inhibition of adherence of zymosan was comparable with that seen with virulent strains of M. tuberculosis in these studies. Down-modulation of macrophage MR also inhibits adherence of Erdman (48 +/- 9%) and H37Rv (20 +/- 2%) in the presence of serum. Simultaneous blockade of MR and CR does not further inhibit adherence of the virulent M. tuberculosis strains over that seen with blocking CR alone.(ABSTRACT TRUNCATED AT 400 WORDS)

1991

Schlesinger, L S, and M A Horwitz. (1991) 1991. “Phenolic Glycolipid-1 of Mycobacterium Leprae Binds Complement Component C3 in Serum and Mediates Phagocytosis by Human Monocytes.”. The Journal of Experimental Medicine 174 (5): 1031-8.

Previous studies from this laboratory have demonstrated that Mycobacterium leprae, an obligate intracellular bacterial parasite, enters human mononuclear phagocytes via complement receptors on these host cells and bacterium-bound C3. The present study investigates the role of M. leprae surface molecules in C3 fixation and phagocytosis. By enzyme-linked immunosorbent assay, C3 binds selectively to phenolic glycolipid-1 (PGL-1), a major surface molecule of the leprosy bacillus. C3 fixation to PGL-1 is serum concentration dependent and is abolished in heat-inactivated serum or serum containing ethylenediaminetetraacetic acid. C3 fixation is also abolished in serum containing ethyleneglycol-bis (beta-aminoethyl ether)N,N,N'-tetraacetic acid and MgCl2 indicating that isolated PGL-1 fixes C3 via the classical complement pathway. The capacity of PGL-1 to fix C3 is dependent upon its terminal trisaccharide since sequential removal of monosaccharide units of the trisaccharide results in a stepwise reduction in C3 fixation. Deacylation of PGL-1 also abolishes C3 fixation. C3 fixes to the trisaccharide of PGL-1 that is chemically linked to bovine serum albumin via the chemical carrier, 8-methoxycarbonyloctanol. PGL-1 mediates C3 fixation to polystyrene microspheres, and PGL-1 and C3 together mediate ingestion of polystyrene microspheres by human monocytes, wherein these inert test particles reside in membrane-bound phagosomes. Thus, complement receptors on mononuclear phagocytes, complement component C3, and PGL-1 comprise a three-component receptor-ligand-acceptor molecule system for mediating phagocytosis of M. leprae.