Publications

In high concentrations of fresh nonimmune human serum, Mycobacterium tuberculosis activates the alternative pathway of complement and binds C3 protein, resulting in enhanced phagocytosis by complement receptors on human alveolar macrophages. Yet in the lung, the alternative pathway of complement is relatively inactive compared to the classical pathway. To begin to determine whether C3 opsonophagocytosis of M. tuberculosis by alveolar macrophages can occur in the lung of the immunologically naive host, we characterized the binding of C3 to M. tuberculosis in different concentrations of fresh nonimmune human serum and concentrated human bronchoalveolar lavage fluid. Here we show that in human serum, C3 binding to M. tuberculosis is rapid, initiated by either the alternative pathway or the classical pathway, depending on the concentration of serum, and occurs by covalent linkages between the bacterial surface and the C3 cleavage products, C3b or C3bi. Human bronchoalveolar lavage fluid contains C3 protein and functional classical pathway activity that mediates the binding of C3 to the surface of M. tuberculosis. These studies provide evidence that when M. tuberculosis is first inhaled into the lungs of the human host, the bacterium is opsonized by C3 cleavage via classical pathway activation within the alveolus, providing a C3-dependent entry pathway into resident alveolar macrophages.

Alveolar macrophages are important host defense cells in the human lung that continuously phagocytose environmental and infectious particles that invade the alveolar space. Alveolar macrophages are prototypical alternatively activated macrophages, with up-regulated innate immune receptor expression, down-regulated costimulatory molecule expression, and limited production of reactive oxygen intermediates (ROI) in response to stimuli. Surfactant protein A (SP-A) is an abundant protein in pulmonary surfactant that has been shown to alter several macrophage (Mphi) immune functions. Data regarding SP-A effects on ROI production are contradictory, and lacking with regard to human Mphi. In this study, we examined the effects of SP-A on the oxidative response of human Mphi to particulate and soluble stimuli using fluorescent and biochemical assays, as well as electron paramagnetic resonance spectroscopy. SP-A significantly reduced Mphi superoxide production in response to the phorbol ester PMA and to serum-opsonized zymosan (OpZy), independent of any effect by SP-A on zymosan phagocytosis. SP-A was not found to scavenge superoxide. We measured Mphi oxygen consumption in response to stimuli using a new oxygen-sensitive electron paramagnetic resonance probe to determine the effects of SP-A on NADPH oxidase activity. SP-A significantly decreased Mphi oxygen consumption in response to PMA and OpZy. Additionally, SP-A reduced the association of NADPH oxidase component p47(phox) with OpZy phagosomes as determined by confocal microscopy, suggesting that SP-A inhibits NADPH oxidase activity by altering oxidase assembly on phagosomal membranes. These data support an anti-inflammatory role for SP-A in pulmonary homeostasis by inhibiting Mphi production of ROI through a reduction in NADPH oxidase activity.

We have reported that Mycobacterium tuberculosis residing within the phagosomes of human monocyte-derived macrophages (MDM) can acquire Fe from extracellular transferrin (TF) and sources within the MDM. In the lung, Fe is also bound to lactoferrin (LF) and low-molecular-weight chelates. We therefore investigated the ability of intraphagosomal M. tuberculosis to acquire Fe from these sources. M. tuberculosis acquired 30-fold and 3-fold more Fe from LF and citrate, respectively, compared to TF, in spite of similar MDM-associated Fe. M. tuberculosis infection decreased MDM-associated Fe relative to uninfected MDM as follows: TF (38.7%), citrate (21.1%), and LF (15.3%). M. tuberculosis Fe acquisition from extracellular chelates (exogenous source) and from endogenous MDM Fe initially acquired from the three chelates (endogenous source) was compared. M. tuberculosis Fe acquisition was similar from exogenous and endogenous sources supplied as Fe-TF. In contrast, there was much greater intracellular M. tuberculosis Fe uptake from LF and citrate from the exogenous than endogenous source. Gamma interferon (IFN-gamma) reduced MDM Fe uptake from each chelate by approximately 50% and augmented the M. tuberculosis-induced decrease in MDM Fe uptake from exogenous TF, but not from LF or citrate. IFN-gamma minimally decreased intracellular M. tuberculosis Fe acquisition from exogenous Fe-TF but significantly increased Fe uptake from LF and citrate. Intraphagosomal M. tuberculosis Fe acquisition from both exogenous and endogenous MDM sources, and the effect of IFN-gamma on this process, is influenced by the nature of the extracellular Fe chelate. M. tuberculosis has developed efficient mechanisms of acquiring Fe from a variety of Fe chelates that it likely encounters within the human lung.

The entry of Mycobacterium tuberculosis (Mtb) into the host macrophage and its survival in this environment are key components of tuberculosis pathogenesis. Following intracellular replication of the bacterium within alveolar macrophages, there is spread of bacilli to regional lymph nodes in the lungs and subsequent presentation of antigens to the host immune system. How this process occurs remains poorly understood, but one mechanism may involve the migration of macrophages containing Mtb across the alveoli to lymph nodes, where there is development of a protective host response with formation of granulomas composed in part of aggregated and fused, apoptotic, infected macrophages. Leukocyte integrins, including lymphocyte function-associated antigen-1 (LFA-1) and complement receptors CR3 and CR4, and their counter receptors play a major role in macrophage adhesion processes and phagocytosis. In this study, the appearance of Mtb-infected macrophages over time was examined, using inverted-phase microscopy and an in vitro culture model of human monocyte-derived macrophages (MDMs). Prior to and immediately following infection of the MDMs with Mtb, the macrophages appeared as individual cells in monolayer culture; however, within 24 h of infection with Mtb, the MDMs began to migrate and adhere to each other. The kinetics of this response were dependent on both the m.o.i. and the length of infection. Quantitative transmission electron microscopy studies revealed that macrophage adhesion was accompanied by increases in levels of LFA-1 and its counter receptor (ICAM-1), decreases in surface levels of the phagocytic receptors CR3, CR4 and FcgammaRII, and an increase in major histocompatibility complex Class II (MHC-II) molecules at 72 h post-infection. Decreases in surface levels of CR3 and CR4 had a functional correlate, with macrophages containing live bacilli showing a diminished phagocytic capacity for complement-opsonized sheep erythrocytes; macrophages containing heat-killed bacilli did not show this diminished capacity. The modulation of macrophage adhesion and phagocytic proteins may influence the trafficking of Mtb-infected macrophages within the host, with increases in levels of LFA-1 and ICAM-1 enhancing the adhesive properties of the macrophage and decreases in phagocytic receptors diminishing the phagocytic capacity of an already-infected cell, potentially allowing for maintenance of the intracellular niche of Mtb.

Cryptococcosis is a leading cause of death among individuals with compromised T cell function. Soluble Cryptococcus neoformans mannoproteins (MP) have emerged as promising vaccine candidates due to their capacity to elicit delayed-type hypersensitivity and Th type 1-like cytokines, both critical to the clearance of this pathogenic yeast. In this study, the mechanisms responsible for the potent immunostimulatory properties of MP were explored. Using Chinese hamster ovary cells expressing human macrophage mannose receptor (MMR), we determined that MP is a MMR ligand. Functionally, competitive blockade of multilectin mannose receptors (MR) on APCs diminished MP-dependent stimulation of primary T cells from immunized mice and the MP-reactive CD4(+) T cell hybridoma, P1D6, by 72 and 99%, respectively. Removal of O-linked saccharides from MP by beta-elimination inhibited MP-dependent stimulation of P1D6 and primary T cells by 89 and 90%, respectively. In addition, MP-dependent stimulation of P1D6 was abrogated after digestion with proteinase K, suggesting the protein core of MP contributed the antigenic moiety presented by APC. Stimulation of P1D6 by MP also was abolished using APC obtained from invariant chain-deficient mice, demonstrating Ag presentation was MHC class II restricted. Our data suggest that MP is a ligand for the MMR and that T cell stimulation is functionally inhibited either by competitive blockade of MR or by removal of carbohydrate residues critical for recognition. The demonstration that efficient T cell responses to MP require recognition of terminal mannose groups by MMR provides both a molecular basis for the immunogenicity of cryptococcal MP and support for vaccination strategies that target MR.

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that "shape" AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A(-/-) mice have reduced MR expression relative to SP-A(+/+). SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.

Mycobacterium tuberculosis multiplies within the macrophage phagosome and requires iron for growth. We examined the route(s) by which intracellular M. tuberculosis acquires iron. During intracellular growth of the virulent Erdman M. tuberculosis strain in human monocyte-derived macrophages (MDM), M. tuberculosis acquisition of (59)Fe from transferrin (TF) provided extracellularly (exogenous source) was compared with acquisition when MDM were loaded with (59)Fe from TF prior to M. tuberculosis infection (endogenous sources). M. tuberculosis (59)Fe acquisition required viable bacteria and was similar from exogenous and endogenous sources at 24 h and greater from exogenous iron at 48 h. Interferon-gamma treatment of MDM reduced (59)Fe uptake from TF 51% and TF receptor expression by 34%. Despite this, intraphagosomal M. tuberculosis iron acquisition in IFN-gamma-treated cells was decreased by only 30%. Macrophages from hereditary hemochromatosis patients have altered iron metabolism. Intracellular M. tuberculosis acquired markedly less iron in MDM from these individuals than in MDM from healthy donors, regardless of the iron source (exogenous and endogenous): 36 +/- 3.8% and 17 +/- 9.6% of control, respectively. Thus, intraphagosomal M. tuberculosis can acquire iron from both extracellular TF and endogenous macrophage sources. Acquisition of iron from macrophage cytoplasmic iron pools may be critical for the intracellular growth of M. tuberculosis. This acquisition is altered by IFN-gamma treatment to a small extent, but is markedly reduced in macrophages from hemochromatosis patients.

The innate immune system in the lung is essential for controlling infections due to inhaled pathogens. Mycobacterium tuberculosis (M.tb) encounters components of the innate immune system when inhaled into the lung, but the consequences of these interactions are poorly understood. Surfactant protein D (SP-D) binds to and agglutinates M.tb bacilli, and reduces the uptake of the bacteria by human macrophages. In the current studies, we utilized a recombinant SP-D variant (CDM) that lacks the collagen domain to further characterize the interaction of SP-D with M.tb, and determine the effects of agglutination on bacterial uptake by human monocyte-derived macrophages. These studies demonstrate that the binding of SP-D and CDM to M.tb is saturable and inhibited by carbohydrate competition and Ca(2+) chelation, implicating the carbohydrate recognition domain in the interaction. Fluorescence microscopy reveals that dodecameric SP-D leads to agglutination of the bacilli, whereas the trimeric CDM does not, demonstrating that the multivalent nature of SP-D is essential for agglutination of M.tb. However, preincubation of M.tb with increasing concentrations of SP-D or CDM leads to a concentration-dependent reduction in the uptake of the bacteria by macrophages, indicating that agglutination does not play a direct role in this observation. Finally, the reduced uptake of M.tb by SP-D is associated with reduced growth of M.tb in monocyte-derived macrophages. These studies provide direct evidence that the inhibition of phagocytosis of M.tb effected by SP-D occurs independently of the aggregation process.