Jon S. Friedland

Mycobacterial 65‐kD heat shock protein induces release of proinflammatory cytokines from human monocytic cells

Monocytes having phagocytosed mycobacteria are known to present the bacterial 65‐kD heat shock protein (hsp) on their cell surface to αβ and γδ T lymphocytes. Cytotoxic CD4+ cells may then lyse monocytes expressing mycobacterial 65‐kD hsp. However, it is not known whether 65‐kD hsp directly stimulates monocyte functions other than antigen presentation. This study has demonstrated that following extraction of bacterial lipopolysaccharide, purified recombinant mycobacterial 65‐kD hsp may directly activate THP‐1 cells, a human monocytic line, to accumulate mRNA for and secrete tumour necrosis factor (TNF), a cytokine important in granuloma formation, the characteristic host immune response to mycobacterial infection. TNF gene expression and secretion following stimulation by hsp was dose‐dependent and abolished by heat‐induced proteolysis. Subsequently, THP‐1 cells secreted IL‐6 and IL‐8, cytokines involved in recruitment and differentiation of T lymphocytes. The data indicate that secretion of proinflammatory cytokines from monocytes activated by mycobacterial 65‐kD hsp may be important in the host immune response and in the development of antigen‐specific T cell‐mediated immunity.

Inhibition of ex vivo proinflammatory cytokine secretion in fatal Mycobacterium tuberculosis infection

Tuberculosis is characterized by fever, weight loss, a prolonged acute‐phase protein response and granuloma formation. These characteristics may partly be due to action of proinflammatory cytokines tumour necrosis factor (TNF), IL‐6 and IL‐8. We investigated plasma concentrations of these cytokines before and after ex vivo lipopolysaccharide stimulation of whole blood leucocytes from 41 Zambian patients with tuberculosis, 32 of whom were also HIV+. Although patients had a reduced weight, were more anaemic and had higher erythrocyte sedimentation rate compared with controls (all P < 0·0005), clinical and laboratory measurements of disease state were similar in those who died and survivors. In contrast, plasma IL‐6 and IL‐8 concentrations were higher in patients who died (P < 0·05). There was no detectable cytokine mRNA in unstimulated leucocytes. There was reduced secretion of TNF (P < 0·005 at 2h), IL‐6 (P < 0·005 at 8 h) and IL‐8 (P < 0·005 at 24 h) after ex vivo stimulation of whole blood leucocytes from patients who died compared with survivors. This was partly due to a soluble inhibitory factor present in plasma. The only additional effect of concurrent infection by HIV with Myco. tuberculosis was decreased IL‐6 secretion following ex vivo stimulation of leucocytes. Reduced proinflammatory cytokine release may represent a critical impairment of host immune defences important in determining outcome in tuberculosis.

Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells

Macrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetyltransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-kappa B binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.

Interleukin-8 and Plasmodium falciparum malaria in Thailand

Jon S. Friedland’, May H02,3, Daniel G. Remick”, llyl;;i Bunnagz, Nicholas J. White*,5 and George E. * 1 ‘Division of Communicable Diseases, St George’s Hospital Medical School, London, UK; *Hospital for Tropical Diseases and Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 3Department of Microbiology and Infectious Diseases, University of Calgay, Calgay, Canada; 4Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA; 5Nuffield Department of Clinical Medicine,John Radcliffe Hospital, Oxford, UK

Phagocytosis of mycobacterium tuberculosis or particulate stimuli by human monocytic cells induces equivalent monocyte chemotactic protein-1 gene expression

Phagocytosis of Mycobacterium tuberculosis by human monocytes or macrophages is classically followed by granuloma formation in vivo. Granuloma are comprised of cells of the monocyte lineage together, in many instances, with antigen-specific T lymphocytes. Development of granuloma depends upon recruitment of both cell types, but recruitment of monocytes is pivotal as these cells secrete anti-mycobacterial cytokines and IL-8, a T cell chemoattractant. We have therefore investigated gene regulation of Monocyte Chemotactic Protein 1 (MCP-1), an important monocyte chemotactic cytokine, following phagocytosis of particulate material (latex beads and zymosan) and live M. tuberculosis by two human monocytic cell lines. In THP-1 cells and phenotypically more differentiated Mono Mac 6 cells, MCP-1 mRNA accumulation was first detectable by Northern analysis of 4 hours and increased over 24 hours. Magnitude and kinetics of MCP-1 gene expression was independent of the biochemical nature of the phagocytic stimulus, M. tuberculosis strain virulence or pre-treatment with anti-TNF. In contrast to the uniform effect of different phagocytic stimuli on MCP-1 gene expression, we have shown that M. tuberculosis but not latex or zymosan, increased IL-8 gene expression, a chemotactic agent for T cells. In additional experiments with THP-1 cells infected with human immunodeficiency virus (HIV), viral infection did not alter MCP-1 gene expression following phagocytosis. MCP-1 gene expression appears to be a conserved antigen-independent response of human monocytic cells which is activated following particulate phagocytosis. MCP-1 gene expression may thus be involved in recruitment of monocytes during granuloma formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential cytokine gene expression and secretion after phagocytosis by a human monocytic cell line of Toxoplasma gondii compared with Mycobacterium tuberculosis.

Toxoplasma gondii mfcclion may be clinically silent in immunocompetent individuals but may cause fatal disease in immunocotripromised patients such as those with HIV infection. Proinnammatory cytokines are known to be important in murine resistance to T. gondii but there are no data from human models of infection. We have investigated whether phagocytosis of T. gondii, of Mycobactcrium tuberculosis (a pathogen which elicits a granulomatous host immune response) and of men latex particles by THP‐I celts, a human monocytic litie. caused gene expression and secretion of tumour neerosis factor (TNF), IL‐6and IL‐8. These cytokines are important in recruitment and activation of T lymphocytes, and both TNF and IL‐6 may have direct antitoxoplasmaeidal and antimycobaeterial activity. Phagocytosis of T. gondii by THP‐1 cells resulted in minimal gene expression and secretion of TNF, IL‐6 and IL‐8 similar to that following phagocytosis of inert latex particles. In contrast, phagocytosis of M. tuberculosis resulted in increased gene expression of TNF and IL‐8 as well as increased secretion of all three cytokines. particularly IL‐8. These observations may partially explain the frequency of non‐inflammatory host responses to T. gondii in immunccompetent individuals.

Modulation of HIV transcription in and release from human monocytic cells following phagocytosis of Mycobacterium tuberculosis

The consequence of phagocytosis of Mycobacterium tuberculosis by human monocytic cells on transcription and release of human immunodeficiency virus (HIV) is unknown. In order to investigate the effects of phagocytosis of M. tuberculosis on HIV transcription, human monocytic THP-1 cells were transfected with constructs of the long terminal repeat of HIV1 or 2 linked to the chloramphenicol acetyl transferase gene (HIV LTR CAT). Following phagocytosis of M. tuberculosis by THP-1 cells maintained in non-adherent conditions, there was enhanced transcription of HIV LTR CAT. This enhancement was further potentiated when such THP-1 cells adhered to tissue culture plastic. Phagocytosis of M. tuberculosis by HIV-infected THP-1 cells in non-adherent conditions had no effect on intact HIV release. However in adherent conditions, phagocytosis of M. tuberculosis reduced release of intact virus even in the face of enhanced HIV transcription. Phagocytosis of M. tuberculosis by THP-1 cells affects HIV replication at both the transcriptional level (upregulates) and the level of viral release (down-regulates) and is modified by cellular adhesion.