Aberrantly low transferrin receptor expression on human monocytes is associated with nonpermissiveness for Legionella pneumophila growth.

Growth of Legionella pneumophila within human monocytes is iron dependent. A person with monocytes uniquely nonpermissive to L. pneumophila growth was identified whose monocytes expressed an abnormally low number of transferrin receptors in the nonactivated state, similar to the typically low level expressed in the interferon-gamma-activated state. The monocytes failed to up-regulate transferrin receptor expression appropriately in response to iron-transferrin. After treatment for chronic periodontal disease, the subject's monocytes converted to a permissive state. In contrast to the nonpermissive state, the permissive monocytes had normal transferrin receptor expression and up-regulated transferrin receptor expression appropriately in response to iron-transferrin. Thus, a nonpermissive state for L. pneumophila intracellular multiplication is associated with low levels of transferrin receptor expression in nonactivated monocytes and with an inability to up-regulate transferrin receptor expression in response to iron-transferrin. This nonpermissive state may be related to chronic inflammatory conditions such as periodontal disease.

The rapidly growing mycobacteria: saprophytes and parasites.

Rapidly growing mycobacteria are widespread saprophytes, but approximately one-third of identified species are also opportunistic pathogens in humans and animals, associated with skin, soft tissue, bone, and pulmonary infections as well as disseminated disease. Clinical and experimental evidence indicates a major role for the cell-mediated immune response in the pathogenesis of infection.

Preliminary characterization of a Mycobacterium abscessus mutant in human and murine models of infection.

The ability to persist in the host after the establishment of infection is an important virulence determinant for mycobacteria. Mycobacterium abscessus is a rapidly growing mycobacterial species which causes a variety of clinical syndromes in humans. We have obtained a rough, wild-type human clinical isolate of M. abscessus (M. abscessus-R) and a smooth, attenuated mutant (M. abscessus-S) which spontaneously dissociated from the clinical isolate. We have found that M. abscessus-R is able to persist and multiply in a murine pulmonary infection model in contrast to M. abscessus-S, which is rapidly cleared. To understand the basis for this difference, we characterized the behavior of these variants in human tissue culture models of infection. M. abscessus-R is able to persist and multiply in human monocytes, while M. abscessus-S is deficient in this ability. Both of these variants are phagocytized by human monocytes. M. abscessus-R resides in a phagosome typical for pathogenic mycobacteria with a tightly adherent phagosomal membrane. In contrast, M. abscessus-S resides in a "loose" phagosome with the phagosomal membrane separated from the bacterial cell wall. Both M. abscessus variants also have distinctive growth patterns in a recently described fibroblast-mycobacterium microcolony assay, with M. abscessus-R exhibiting growth characteristics similar to those previously reported for virulent M. tuberculosis and M. abscessus-S exhibiting growth characteristics similar to those previously reported for avirulent M. tuberculosis. In both the monocyte infection assay and the murine pulmonary infection model, numerous infected mononuclear phagocyte aggregates develop at sites of M. abscessus-R infection, but are absent with M. abscessus-S infection. We conclude that a mutation has occurred in the M. abscessus-S variant which has altered the ability of this organism to persist and multiply in host cells and that this may be related to the phenotypic changes we have observed in our tissue culture models of infection.

Differential growth characteristics and streptomycin susceptibility of virulent and avirulent Mycobacterium tuberculosis strains in a novel fibroblast-mycobacterium microcolony assay.

The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 microgram/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 microgram/ml. Growth of all strains was similarly inhibited by 1.0 microgram of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.

Multinucleated giant cell formation induced by IFN-gamma/IL-3 is associated with restriction of virulent Mycobacterium tuberculosis cell to cell invasion in human monocyte monolayers.

One of the hallmarks of an effective immune response against Mycobacterium tuberculosis is the formation of granulomas containing multinucleated giant cells. IFN-gamma and interleukin-3 (IL-3) promote Langhans-type multinucleated giant cell formation and have been identified in T cell clones reacting to M. tuberculosis antigens. The ability of human monocytes treated with IFN-gamma and IL-3 to limit the spread of M. tuberculosis in an in vitro infection assay was examined. Monocytes were incubated with control medium, IFN-gamma, TNF-alpha, and calcitriol, a combination permissive to M. tuberculosis growth, or IFN-gamma and IL-3 and infected with a low inoculum of M. tuberculosis (Erdman). IFN-gamma/IL-3 treatment reduced M. tuberculosis CFU relative to both untreated and IFN-gamma/TNF-alpha/calcitriol-treated monocytes. Specifically, CFU were reduced by 79% at 14 days in the IFN-gamma/IL-3 treatment group relative to the IFN-gamma/TNF-alpha/calcitriol treatment group, an effect that was not due to toxic monocyte metabolites. M. tuberculosis growth restriction by IFN-gamma/IL-3-treated monocyte monolayers was associated with the development of Langhans-type multinucleated giant cells. At the light microscope level, dense growth of M. tuberculosis surrounded by a ring of nuclei localized to the center of individual cells. The intracellular location of M. tuberculosis was confirmed by electron microscopy. In contrast, monocyte monolayers treated with IFN-gamma/TNF-alpha/calcitriol consisted of a syncitium of cells containing monocyte aggregates. Nonlocalized linear arrays of M. tuberculosis were observed to be growing throughout such aggregates. These results suggest that physical sequestration of M. tuberculosis by Langhans-type multinucleated giant cells may limit cell to cell spread of this pathogen, thereby restricting growth.

Tumor necrosis factor alpha (TNFalpha) promotes growth of virulent Mycobacterium tuberculosis in human monocytes iron-mediated growth suppression is correlated with decreased release of TNFalpha from iron-treated infected monocytes.

The human immune response to Mycobacterium tuberculosis is not well characterized. To better understand the cellular immune response to tuberculosis, a human mononuclear phagocyte culture system using a low-infecting inoculum of M. tuberculosis to mimic in vivo conditions was developed. Using this system, monocytes treated with IFNgamma/TNFalpha/ calcitriol (CytD) were permissive for the growth of virulent M. tuberculosis. In the presence of iron, however, these monocytes suppressed the growth of M. tuberculosis. The enhanced permissiveness of CytD-preincubated monocytes was found to be due to TNFalpha, however, the ability of iron to suppress M. tuberculosis growth also required preincubation with TNFalpha. Iron-mediated growth suppression was correlated with selective suppression of TNFalpha release from infected monocytes. In addition, removal of TNFalpha from CytD-treated monocytes 2 d after infection mimicked the suppressive effect of iron, suggesting that iron may also be decreasing monocyte sensitivity to exogenously added TNFalpha. In the absence of iron, permissive, CytD-treated monocytes formed large infected cellular aggregates. With iron treatment, aggregation was suppressed, suggesting that the iron-suppressive effect on M. tuberculosis growth may be related to suppression of monocyte aggregation and diminished cell-to-cell spread of M. tuberculosis. The results of this study indicate that TNFalpha preincubation is required for human monocytes to exert an iron-mediated suppressive effect on M. tuberculosis growth. In the absence of iron, however, the continued presence of TNFalpha has a growth-promoting effect on M. tuberculosis in human monocytes. Iron may be an important early modulator of M. tuberculosis growth via its effects on TNFalpha.

Cutaneous miliary tuberculosis in the AIDS era: case report and review.

Tuberculosis with extrapulmonary manifestations is common in patients with AIDS. The skin is a site of dissemination that has often been overlooked. Historically, cutaneous miliary tuberculosis, also known as tuberculosis cutis miliaris disseminata, was noted to be a rare entity in adults; however, over the past 5 years, five cases of cutaneous miliary tuberculosis in human immunodeficiency virus (HIV)-seropositive individuals have been reported. We present the sixth such case and review the medical literature on cutaneous miliary tuberculosis in adults both before and during the AIDS era. The incidence of tuberculosis cutis miliaris disseminata in HIV-seropositive adults is likely higher than the incidence among the HIV-seronegative population that has been suggested in the historical literature. Its appearance can be quite nondescript; a high index of suspicion must be maintained, particularly for those patients with a CD4 cell count of < 200/mm3, to achieve the proper diagnosis and initiate appropriate therapy.

Cytokines and legionellosis.

Recent studies have led to an enhanced understanding of the role of cell-mediated immunity and cytokines in Legionnaires' disease. In particular, the effect of interferon gamma on human mononuclear phagocyte iron metabolism and the role of iron availability of Legionella pneumophila intracellular multiplication in human monocytes has been elucidated. With this knowledge it is now possible to develop treatment strategies for Legionnaires' disease using interferon gamma and/or agents affecting human mononuclear phagocyte iron metabolism.

Regulation of transferrin receptor expression and ferritin content in human mononuclear phagocytes. Coordinate upregulation by iron transferrin and downregulation by interferon gamma.

We have investigated the regulation of key human iron binding proteins in mononuclear phagocytes by IFN gamma and iron transferrin. In a previous study, we demonstrated that IFN gamma downregulates the expression on human monocytes of transferrin receptors, the major source of iron for the cell. In the present study, we show that IFN gamma also downregulates the intracellular concentration of ferritin, the major iron storage protein in the cell. By radioimmunoassay, the mean ferritin content of nonactivated monocytes was 361 +/- 107 fg/monocyte (mean +/- SEM) whereas the mean ferritin content of IFN gamma-activated monocytes was 64 +/- 13 fg/monocyte, an 82% reduction with activation (P < 0.01, t test). Consistent with its downregulating effect on these iron proteins, IFN gamma treatment also results in decreased iron incorporation. IFN gamma-activated monocytes incorporated 33% less iron from 59Fe-transferrin than nonactivated monocytes (P < 0.05, t test). Gel filtration chromatography revealed that incorporated iron is located primarily in ferritin in both nonactivated and IFN gamma-activated monocytes. Ferritin in IFN gamma-activated monocytes is saturated with approximately three times as much 59Fe as ferritin in nonactivated monocytes. We have also explored the effect of iron transferrin on transferrin receptor expression and intracellular ferritin content in human monocytes. We have found that iron transferrin markedly upregulates both transferrin receptor expression and intracellular ferritin content in both nonactivated (2.3- and 1.3-fold, respectively) and IFN gamma-activated (3.4- and 2.9-fold, respectively) monocytes. This study demonstrates that transferrin receptor expression and intracellular ferritin content in human monocytes is unidirectionally and coordinately upregulated by iron transferrin and unidirectionally and coordinately downregulated by IFN gamma.

Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila.

We have been exploring the role of iron in the pathogenesis of the intracellular bacterial pathogen Legionella pneumophila. In previous studies, we have demonstrated that L. pneumophila intracellular multiplication in human monocytes is iron dependent and that IFN gamma-activated monocytes inhibit L. pneumophila intracellular multiplication by limiting the availability of iron. In this study, we have investigated the effect on L. pneumophila intracellular multiplication of lactoferrin, an iron-binding protein which is internalized via specific receptors on monocytes, and of nonphysiologic iron chelates which enter monocytes by a receptor-independent route. Apolactoferrin completely inhibited L. pneumophila multiplication in nonactivated monocytes, and enhanced the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication. In contrast, iron-saturated lactoferrin had no effect on the already rapid rate of L. pneumophila multiplication in nonactivated monocytes. Moreover, it reversed the capacity of activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron from the lactoferrin-lactoferrin receptor pathway. The capacity of iron-lactoferrin to reverse monocyte activation was dependent upon its percent iron saturation and not just its total iron content. Similarly, the nonphysiologic iron chelates ferric nitrilotriacetate and ferric ammonium citrate completely reverse and ferric pyrophosphate partially reversed the capacity of IFN gamma-activated monocytes to inhibit L. pneumophila intracellular multiplication, demonstrating that L. pneumophila can utilize iron derived from nonphysiologic iron chelates internalized by monocytes independently of the transferrin and lactoferrin endocytic pathways. This study suggests that at sites of inflammation, lactoferrin may inhibit or promote L. pneumophila intracellular multiplication in mononuclear phagocytes depending upon its degree of iron saturation. In addition, this study suggests a potential role for PMN in host defense against L. pneumophila--providing apolactoferrin to infected monocytes--and it supports the concept that PMN and monocytes may cooperate in host defense against intracellular parasites and other pathogens.

Chloroquine inhibits the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. A potential new mechanism for the therapeutic effect of chloroquine against intracellular pathogens.

Chloroquine and ammonium chloride, by virtue of their basic properties, have been shown to raise endocytic and lysosomal pH and thereby interfere with normal iron metabolism in a variety of cell types, including mononuclear phagocytes. Cellular iron metabolism is of critical importance to Legionella pneumophila, an intracellular bacterial pathogen whose capacity to multiply in human mononuclear phagocytes is dependent upon the availability of intracellular iron. In view of this, we have studied the effects of chloroquine and ammonium chloride on L. pneumophila intracellular multiplication in human monocytes. Chloroquine, at a concentration of 20 microM, and ammonium chloride, at a concentration of 20 mM, inhibited L. pneumophila intracellular multiplication by 1.4 +/- 0.2 (SEM) logs and 1.5 +/- 0.2 logs, respectively. Chloroquine- and ammonium chloride-induced inhibition of L. pneumophila intracellular multiplication was completely reversed by iron nitrilotriacetate, an iron compound which is soluble in the neutral to alkaline pH range, but not by iron transferrin, which depends upon acidic intracellular conditions to release iron. Chloroquine had no major direct effect on L. pneumophila multiplication in artificial media except at extremely high concentrations (15,000-fold that which inhibited L. pneumophila multiplication in mononuclear phagocytes), and inhibition at such concentrations was not reversed by iron nitrilotriacetate. This study demonstrates that chloroquine and ammonium chloride inhibit the intracellular multiplication of L. pneumophila by limiting the availability of iron to the bacterium. It is possible that such a mechanism of action underlies chloroquine's antimicrobial effect against other intracellular pathogens, such as the agents of malaria and tuberculosis.

Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron.

We have investigated the role of iron in the intracellular biology of Legionella pneumophila in human monocytes and in the effector arm of cell-mediated immune defense against this intracellular bacterial pathogen. To determine if L. pneumophila intracellular multiplication is iron dependent, we studied the effect of the iron chelator deferoxamine on L. pneumophila infection of monocytes. Deferoxamine at 15 microM completely inhibited L. pneumophila intracellular multiplication. The inhibitory effect of deferoxamine was reversed with equimolar iron-saturated transferrin but not apotransferrin. To examine the potential role of iron in monocyte activation, we investigated the influence of iron-saturated transferrin on L. pneumophila multiplication in IFN gamma-activated monocytes. Iron transferrin, but not apotransferrin, neutralized the capacity of activated monocytes to inhibit L. pneumophila multiplication. To explore a potential mechanism by which activated monocytes might limit the availability of intracellular iron, we examined transferrin receptor expression on nonactivated and activated monocytes cultured in vitro for 5 d. By fluorescence-activated flow cytometry, activated monocytes exhibited markedly fewer transferrin receptors than nonactivated monocytes. By Scatchard analysis of 125I-transferrin binding to monocytes, nonactivated monocytes had 38,300 +/- 12,700 (mean +/- SE) transferrin binding sites, whereas activated monocytes had 10,300 +/- 1,600, a reduction of 73%. Activated and nonactivated monocytes had a similar mean Kd (1.8 +/- 0.2 nM). This study demonstrates that (a) L. pneumophila intracellular multiplication is iron dependent; (b) activated monocytes inhibit L. pneumophila multiplication by limiting the availability of intracellular iron; and (c) transferrin receptors are downregulated on IFN gamma-activated monocytes.