Changes in the number of alveolar macrophages were correlated with organism burden during Pneumocystis carinii infection. The lungs of healthy, dexamethasone-treated, and dexamethasone-treated and P. cariniiinfected rats were lavaged with phosphate-buffered saline. Counting of alveolar macrophages in the lavage fluids revealed that P. carinii infection caused a 58% decrease in the number of alveolar macrophages and that higher P. carinii organism burdens caused a more rapid decrease in alveolar macrophage number. As a control, healthy rats were challenged with the same number of organisms as that normally used to generate P. carinii infections in dexamethasone-treated rats. Thirteen days after challenge, these rats had a profound (54%) increase in alveolar macrophage number in response to the challenge, while the number of alveolar macrophages in immunosuppressed and P. carinii-infected rats had decreased significantly by this time point. These experiments created the first animal model to mimic human pneumocystis pneumonia in alveolar macrophage number alterations. Reduction of P. carinii organism numbers by treatment of rats with trimethoprim and sulfamethoxazole brought a slow rebound in alveolar macrophage number, while recovery from P. carinii infection by cessation of immunosuppression brought a rapid rebound in alveolar macrophage number. These results suggest that both the immune state of the host and P. carinii burden affect alveolar macrophage number.Immunosuppressed populations are at risk for developing a pneumonia caused by the opportunistic organism Pneumocystis carinii (renamed recently as Pneumocystis jiroveci for those strains that infect humans) (31). P. carinii now refers to those organisms that infect rats (31). Infection with pneumocystis organisms brings many changes in the lung environment, including decrease of phosphatidylcholine (27,29) and increase of sphingomyelin (T. H. Su, V. Natarajan, and W. J. Martin II, abstract from the 1992 Int. Conf. Am. Lung Assoc./Am. Thoracic Soc., Am. Rev. Resp. Dis. 145:A246, 1992), surfactant proteins A and D (22, 26) and the adhesion proteins vitronectin and fibronectin (19). Pneumocystis infection also causes increases in the production of the cytokines tumor necrosis factor alpha (11, 24, 37), interleukin-8 (IL-8), 39), and gamma interferon (39); the growth factor granulocytemacrophage colony-stimulating factor (24); and the chemokine monocyte chemotactic protein 1 (2) but down-regulates the expression of the host transcription factor GATA-2 (33). An inflammatory response (41, 43) that changes alveolar macrophage and polymorphonuclear cell populations (7,42,44) is also observed during pneumocystis infection.In immunocompetent humans and animals, alveolar macrophages protect against pneumocystis infection by actively removing the organism from the alveolus. In contrast, it has been shown that alveolar macrophages from pneumocystis-infected animals do not phagocytose pneumocystis organisms to any significant degree (3,13). In addition to the defect in ...

The innate immune response to Pneumocystis infection is not well understood. In this study, normal C57BL/6 mouse alveolar macrophages were found to respond to Pneumocystis murina organisms through Toll-like receptor 2 (TLR2), leading to the nuclear translocation of NF-B and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-␣) and chemokine macrophage inflammatory protein 2 (MIP-2). P. murina stimulation of normal alveolar macrophages from C57BL/6 mice resulted in increased TLR2 transcription but not increased TLR4 transcription. In gain-of-function studies with HEK293 cells expressing TLR2 or TLR4, only TLR2 was found to stimulate an NF-B response to P. murina. TNF-␣ and MIP-2 production in response to P. murina by mouse alveolar macrophages was inhibited by a monoclonal antibody that specifically blocked the ligand-binding ability of TLR2. Alveolar macrophages from TLR2 knockout (TLR2 ؊/؊ ) mice showed little increase in TNF-␣ and MIP-2 mRNA levels upon P. murina stimulation. An in vivo study showed that TLR2 ؊/؊ mice challenged with P. murina had reduced cytokine responses. These results indicate that TLR2 plays a major role in the innate immune response to P. murina.Pneumocystis is an opportunistic fungal pathogen of immunocompromised hosts. Pneumocystis organisms that infect humans are now called Pneumocystis jiroveci (41) and are a common cause of pneumonia in patients with AIDS (37). Pneumocystis carinii and Pneumocystis murina are those species that infect rats and mice, respectively (17,41). Although the use of highly active antiretroviral therapy has decreased the incidence of Pneumocystis pneumonia (PcP), the morbidity and mortality rates of PcP remain high (30). Patients who receive long-term immunosuppressive medications or who have genetic immunodeficiency are also susceptible to Pneumocystis infection.Effective immune responses of the host are required to control PcP. Alveolar macrophages play a critical role in the innate immune response to many microbes. Experimental evidence indicates that alveolar macrophages contribute to the host immune response to Pneumocystis organisms by directly ingesting or killing them and producing inflammatory cytokines and chemokines such as tumor necrosis factor alpha (TNF-␣) and macrophage inflammatory protein 2 (MIP-2) (14, 15, 54). The exact mechanism by which Pneumocystis stimulates alveolar macrophages to produce cytokines and chemokines is largely unknown.Microbes induce immune responses of host cells by interacting with certain cell surface receptors, termed pattern recognition receptors. Toll-like receptors (TLRs) have been identified as important pattern recognition receptors (28, 34) and are a large family with at least 11 members. Each TLR has a specific ligand(s) (44). TLR4 and its coreceptor, MD2, recognize lipopolysaccharides (LPSs) from gram-negative bacteria as well as many other ligands such as glucuronoxylomannan found in the polysaccharide capsule of Cryptococcus neoformans (38) and glycoinositolphospholipids from Trypano...

The number of alveolar macrophages is decreased during Pneumocystis pneumonia (Pcp), partly because of activation of apoptosis in these cells. This apoptosis occurs in both rat and mouse models of Pcp. Bronchoalveolar lavage (BAL) fluids from Pneumocystis-infected animals were found to contain high levels of polyamines, including spermidine, N 1 -acetylspermine, and N 1 -acetylspermidine. These BAL fluids and exogenous polyamines were able to induce apoptosis in alveolar macrophages. Apoptosis of alveolar macrophages during infection, after incubation with BAL fluids from Pneumocystis-infected animals, or after incubation with polyamines was marked by an increase in intracellular reactive oxygen species, activation of caspases-3 and -9, DNA fragmentation, and leakage of mitochondrial cytochrome c into the cytoplasm. When polyamines were depleted from the BAL fluids of infected animals, the ability of these BAL fluids to induce apoptosis was lost. Interestingly, the apoptosis inducing activity of the polyamine-depleted BAL fluids was restored when polyamines were added back. The results of this study suggested that Pneumocystis infection results in accumulation of high levels of polyamines in the lung. These polyamines activate apoptosis of alveolar macrophages, perhaps because of the ROS that are produced during polyamine metabolism.Pneumocystis-infected lungs usually contain much alveolar exudate and numerous inflammatory cells in perivascular and peribronchiolar areas (1-3). The infection also causes changes in the lung. One such change is significantly increased levels of surfactant proteins A and D (4, 5). These collectins are implicated in the attachment of Pneumocystis to alveolar epithelial cells during infection (4 -6) and evasion of the host immune response (7). In contrast, surfactant proteins B (8) and C (4, 9) are down-regulated in their expression. Macrophage mannose receptor expression is also down-regulated (10), although the shed form of the mannose receptor is increased (11), which may help the organism evade host immune responses (7,10,11). The expression of the transcription factor GATA-2 is reduced in alveolar macrophages during Pcp 2 (12), and this GATA-2 down-regulation is correlated with the dysfunction of alveolar macrophages (13). There is a decrease in plasma S-adenosylmethionine levels during the infection (14). The infection also causes erosion of type I pneumocytes and proliferation of type II epithelial cells (15). These changes indicate that Pneumocystis mediates many alterations in pulmonary and systemic environments in an effort to survive in the host.In addition to these changes, the number of alveolar macrophages is decreased during Pcp in humans (16 -20) and in a rat model of infection (21). This change may be due to decreased precursor cell recruitment or maturation, increased efflux of cells from the lung, increased apoptosis rate, or a combination of these factors. Apoptosis is a normal event in development or tissue turnover (22) and can also be a response to disease stat...

Experiments were performed to determine whether this defect is specific for P. carinii organisms. The results showed that these macrophages were unable to phagocytose both P. carinii organisms and fluorescein isothiocyanate (FITC)-conjugated latex beads, indicating that alveolar macrophages from P. carinii-infected hosts have a general defect in phagocytosis. To determine whether this defect correlates with the recently discovered down-regulation of the GATA-2 transcription factor gene during P. carinii infection, alveolar macrophages from dexamethasone-suppressed or healthy rats were treated with anti-GATA-2 oligonucleotides and then assayed for phagocytosis. Aliquots of the alveolar macrophages were also treated with the sense oligonucleotides as the control. Cells treated with the antisense oligonucleotides were found to have a 46% reduction in phagocytosis of P. carinii organisms and a 65% reduction in phagocytosis of FITC-latex beads compared to those treated with the sense oligonucleotides. To determine whether the defect in phagocytosis in alveolar macrophages from P. carinii-infected hosts can be corrected by overexpression of GATA-2, a plasmid containing the rat GATA-2 gene in the sense orientation driven by the cytomegalovirus (CMV) promoter was introduced into alveolar macrophages from P. carinii-infected rats. Aliquots of the same cells transfected with a plasmid containing GATA-2 in the antisense orientation relative to the CMV promoter served as the control. Alveolar macrophages treated with the sense GATA-2 expression construct were found to increase their phagocytic activity by 66% in phagocytosis of P. carinii organisms and by 280% in phagocytosis of FITC-latex beads compared to those that received the antisense GATA-2 construct. The results of this study indicate that GATA-2 plays an important role in the regulation of phagocytosis in alveolar macrophages during P. carinii infection.

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40–60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.