Role of complement activation in obliterative bronchiolitis post-lung transplantation.

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17-regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1-related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1-related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17-mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.

Defective nitric oxide production by alveolar macrophages during Pneumocystis pneumonia.

The effect of nitric oxide (NO) on Pneumocystis (Pc) organisms, the role of NO in the defense against infection with Pc, and the production of NO by alveolar macrophages (AMs) during Pneumocystis pneumonia (PCP) were investigated. The results indicate that NO was toxic to Pc organisms and inhibited their proliferation in culture. When the production of NO was inhibited by intraperitoneal injection of rats with the nitric oxide synthase inhibitor L-N(5)-(1-iminoethyl) ornithine, progression of Pc infection in immunocompetent rats was enhanced. Concentrations of NO in bronchoalveolar lavage fluids from immunosuppressed, Pc-infected rats and mice were greatly reduced, compared with those from uninfected animals, and AMs from these animals were defective in NO production. However, inducible nitric oxide synthase (iNOS) mRNA and protein concentrations were high in AMs from Pc-infected rats and mice. Immunoblot analysis showed that iNOS in AMs from Pc-infected rats existed primarily as a monomer, but the homo-dimerization of iNOS monomers was required for the production of NO. When iNOS dimerization cofactors, including calmodulin, were added to macrophage lysates, iNOS dimerization increased, whereas incubation of the same lysates with all cofactors except calmodulin did not rescue iNOS dimer formation. These data suggest that NO is important in the defense against Pc infection, but that the production of NO in AMs during PCP is defective because of the reduced dimerization of iNOS.

Downregulation of PU.1 leads to decreased expression of Dectin-1 in alveolar macrophages during Pneumocystis pneumonia.

Dectin-1 is an important macrophage phagocytic receptor recognizing fungal beta-glucans. In this study, the mRNA levels of the Dectin-1 gene were found to be decreased by 61% in alveolar macrophages (AMs) from Pneumocystis-infected mice. The expression of Dectin-1 protein on the surface of these cells was also significantly decreased. By fluorescence in situ hybridization, mRNA expression levels of the transcription factor PU.1 were also found to be significantly reduced in AMs from Pneumocystis-infected mice. Electrophoretic mobility shift assay showed that PU.1 protein bound Dectin-1 gene promoter. With a luciferase reporter gene driven by the Dectin-1 gene promoter, the expression of the PU.1 gene in NIH 3T3 cells was found to enhance the luciferase activity in a dose-dependent manner. PU.1 expression knockdown by small interfering RNA (siRNA) caused a 63% decrease in Dectin-1 mRNA level and 40% decrease in protein level in AMs. Results of this study indicate that downregulation of PU.1 during Pneumocystis pneumonia leads to decreased expression of Dectin-1 in AMs.

Microarray studies on effects of Pneumocystis carinii infection on global gene expression in alveolar macrophages.

BACKGROUND: Pneumocystis pneumonia is a common opportunistic disease in AIDS patients. The alveolar macrophage is an important effector cell in the clearance of Pneumocystis organisms by phagocytosis. However, both the number and phagocytic activity of alveolar macrophages are decreased in Pneumocystis infected hosts. To understand how Pneumocystis inactivates alveolar macrophages, Affymetrix GeneChip RG-U34A DNA microarrays were used to study the difference in global gene expression in alveolar macrophages from uninfected and Pneumocystis carinii-infected Sprague-Dawley rats. RESULTS: Analyses of genes that were affected by Pneumocystis infection showed that many functions in the cells were affected. Antigen presentation, cell-mediated immune response, humoral immune response, and inflammatory response were most severely affected, followed by cellular movement, immune cell trafficking, immunological disease, cell-to-cell signaling and interaction, cell death, organ injury and abnormality, cell signaling, infectious disease, small molecular biochemistry, antimicrobial response, and free radical scavenging. Since rats must be immunosuppressed in order to develop Pneumocystis infection, alveolar macrophages from four rats of the same sex and age that were treated with dexamethasone for the entire eight weeks of the study period were also examined. With a filter of false-discovery rate less than 0.1 and fold change greater than 1.5, 200 genes were found to be up-regulated, and 144 genes were down-regulated by dexamethasone treatment. During Pneumocystis pneumonia, 115 genes were found to be up- and 137 were down-regulated with the same filtering criteria. The top ten genes up-regulated by Pneumocystis infection were Cxcl10, Spp1, S100A9, Rsad2, S100A8, Nos2, RT1-Bb, Lcn2, RT1-Db1, and Srgn with fold changes ranging between 12.33 and 5.34; and the top ten down-regulated ones were Lgals1, Psat1, Tbc1d23, Gsta1, Car5b, Xrcc5, Pdlim1, Alcam, Cidea, and Pkib with fold changes ranging between -4.24 and -2.25. CONCLUSIONS: In order to survive in the host, Pneumocystis organisms change the expression profile of alveolar macrophages. Results of this study revealed that Pneumocystis infection affects many cellular functions leading to reduced number and activity of alveolar macrophages during Pneumocystis pneumonia.

Effects of decreased calmodulin protein on the survival mechanisms of alveolar macrophages during Pneumocystis pneumonia.

Pneumocystis infection causes increased intracellular levels of reactive oxygen species (ROS) and the subsequent apoptosis of alveolar macrophages (Amø). Assessments of key prosurvival molecules in Amø and bronchoalveolar lavage fluids from infected rats and mice showed low levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and reduced activation of phosphoinositide-3 kinase (PI-3K). Ubiquitous calcium-sensing protein calmodulin protein and mRNA levels were also reduced in Amø during Pneumocystis pneumonia (Pcp). Calmodulin has been implicated in control of GM-CSF production and PI-3K activation in other immune cell types. Experiments to determine the control of GM-CSF and PI-3K by calmodulin in Amø showed that GM-CSF expression and PI-3K activation could not be induced when calmodulin was inhibited. Calmodulin inhibition also led to increased levels of ROS and apoptosis in cells exposed to bronchoalveolar lavage fluids from infected animals. Supplementation of Amø with exogenous calmodulin increased survival signaling via GM-CSF and PI-3K and reduced ROS and apoptosis. These data support the hypotheses that calmodulin levels at least partially control survival signaling in Amø and that restoration of GM-CSF or PI-3K signaling will improve host response to the organism.

Polyamine transport as a target for treatment of Pneumocystis pneumonia.

Polyamine levels are greatly increased in alveolar macrophages (AMs) during Pneumocystis pneumonia (PCP), leading to increased production of H(2)O(2), which causes AMs to undergo apoptosis. One of the mechanisms by which polyamine levels in AMs are elevated is enhanced uptake of exogenous polyamines. In this study, the possibility of targeting polyamine uptake as a treatment for PCP was examined. Four anthracene- and one benzene-polyamine conjugates that are potential polyamine transport inhibitors, including N1-anthracen-9-ylmethyl-butane-1,4-diamine; N-(4-aminobutyl)-N-anthracen-9-ylmethylbutane-1,4-diamine; N-[4-(4-aminobutylamino)butyl]-N-anthracen-9-ylmethylbutane-1,4-diamine; N-(4-amino-butyl)-N'-(10-[[4-(4-amino-butylamino)butylamino]-methyl]anthracen-9-ylmethyl)butane-1,4-diamine (44-Ant-44); and benzene-polyamine conjugate N-(4-amino-butyl)-N'-(4-[[4-(4-amino-butylamino)butylamino]-methyl]benzyl)butane-1,4-diamine (44-Bn-44), were tested. Compounds 44-Ant-44 and 44-Bn-44 were found to have a very low toxicity to AMs in vitro and were evaluated for their therapeutic effect on PCP in vivo. Sprague-Dawley rats infected with P. carinii for 28 days were intranasally instilled with 50 microl of a 1 mM solution of 44-Bn-44 or 44-Ant-44 every 2 days. Twenty-one days after initiation of the treatment, three to five rats from each group were sacrificed and examined for lung pathology, organism burden, and apoptosis of AMs. Both 44-Bn-44 and 44-Ant-44 reduced organism burdens; however, only 44-Ant-44 decreased the severity of the infection with reduced lung inflammation, increased clearance of exudates, increased air space, and decreased apoptosis of AMs. 44-Ant-44 also significantly prolonged the survival of treated animals. These results suggest that polyamine uptake is a potential target for treatment of PCP.

Decreased inflammatory response in Toll-like receptor 2 knockout mice is associated with exacerbated Pneumocystis pneumonia.

Pneumocystis pneumonia (PcP) is marked by substantial inflammatory damage to the lung. We have found that Toll-like receptor 2 (TLR2) mediates macrophage inflammatory responses to Pneumocystis and hypothesized that TLR2 deficiency would lead to less severe inflammation and milder lung injury during PcP. Histopathology examination showed that TLR2-/- mice with PcP indeed exhibited milder pulmonary inflammation. TLR2-/- mouse lungs contained less TNF-alpha and displayed lower levels of NF-kappaB activation during PcP. However, TLR2-/- mice with PcP displayed increased severity in symptoms and organism burden. The increased organism burden is likely due to defects in protective mechanisms in TLR2-/- mice. mRNA levels of the inducible nitric oxide synthase and NADPH oxidase p47phox, as well as nitric oxide levels in the lungs, were decreased in TLR2-/- PcP mice. Taken together, this study shows that TLR2-mediated inflammatory responses contribute to a certain degree to the clearance of Pneumocystis organism in mice.

Pneumocystis mediates overexpression of antizyme inhibitor resulting in increased polyamine levels and apoptosis in alveolar macrophages.

Pneumocystis pneumonia (PcP) is the most common opportunistic disease in immunocompromised patients. Alveolar macrophages are responsible for the clearance of Pneumocystis organisms; however, they undergo a high rate of apoptosis during PcP due to increased intracellular polyamine levels. In this study, the sources of polyamines and mechanisms of polyamine increase and polyamine-induced apoptosis were investigated. The level of ornithine decarboxylase (ODC) was elevated in alveolar macrophages, and the number of alveolar macrophages that took up exogenous polyamines was increased 20-fold during PcP. Monocytes, B lymphocytes, and CD8+ T lymphocytes that were recruited into the lung during PcP expressed high levels of ornithine decarboxylase, suggesting that these cells are sources of polyamines. Both protein and mRNA levels of antizyme inhibitor (AZI) were increased in alveolar macrophages during PcP. This AZI overexpression correlated with increased polyamine uptake by alveolar macrophages, because AZI expression knockdown decreased the polyamine uptake ability of these cells. AZI expression knockdown also decreased the apoptosis rate of alveolar macrophages. Pneumocystis organisms and zymosan A were found to induce AZI overexpression in alveolar macrophages, suggesting that beta-glucan, which is the major component of the Pneumocystis cell wall, induces AZI overexpression. The levels of mRNA, protein, and activity of polyamine oxidase were increased in alveolar macrophages during PcP, indicating that the H(2)O(2) generated during polyamine catabolism caused alveolar macrophages to undergo apoptosis. Taken together, results of this study indicate that Pneumocystis organisms induce AZI overexpression in alveolar macrophages, leading to increased polyamine synthesis and uptake and apoptosis rate of these cells.

Polyamine-mediated apoptosis of alveolar macrophages during Pneumocystis pneumonia.

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, N1-acetylspermine, and N1-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.

Suppression of alveolar macrophage apoptosis prolongs survival of rats and mice with pneumocystis pneumonia.

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.

Toll-like receptor 2 mediates alveolar macrophage response to Pneumocystis murina.

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-kappaB and the production of proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) 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-kappaB response to P. murina. TNF-alpha 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-alpha 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.

Effect of bronchoalveolar lavage fluid from Pneumocystis carinii-infected hosts on phagocytic activity of alveolar macrophages.

Alveolar macrophages from Pneumocystis carinii-infected rats are defective in phagocytosis. To investigate whether this defect is due to a certain factor present in P. carinii-infected lungs, alveolar macrophages from uninfected rats were incubated with bronchoalveolar lavage (BAL) fluid samples from P. carinii-infected rats. Alveolar macrophages treated with these BAL fluid samples became defective in phagocytosis but remained normal when treated with BAL fluid samples from noninfected or Toxoplasma gondii-infected rats. The suppressive activity of the BAL fluid samples from P. carinii-infected rats on phagocytosis was retained when the BAL fluid samples were passed through a filter with a pore size of 0.45 microm but was lost when the BAL fluid samples were digested with proteases such as trypsin, pepsin, papain, or endopeptidase Gly-C. Lipid fractions of these BAL fluid samples had no suppressive activity on phagocytosis. The suppressive activity of these BAL fluid samples was also lost when they were incubated with concanavalin A-agarose beads, suggesting that the inhibitor is a glycoprotein. The inhibitor was estimated to be larger than 100,000 Da by exclusion filtration. After binding to the concanavalin A-agarose beads, the inhibitor in BAL fluid samples and P. carinii lysate could be eluted with 200 mM methylmannose. Treatment of both the crude BAL fluid samples and P. carinii lysate and the 200 mM methylmannose eluate with antibody against the major surface glycoprotein of P. carinii eliminated their suppressive activity. These results suggest that the factor capable of suppressing the phagocytic activity of alveolar macrophages is P. carinii major surface glycoprotein or one or more of its derivatives.

Effect of transcription factor GATA-2 on phagocytic activity of alveolar macrophages from Pneumocystis carinii-infected hosts.

Alveolar macrophages from Pneumocystis carinii-infected hosts are defective in phagocytosis (W. Chen, J. W. Mills, and A. G. Harmsen, Int. J. Exp. Pathol. 73:709-720, 1992; H. Koziel et al., J. Clin. Investig. 102:1332-1344, 1998). 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.

Correlation of organism burden and alveolar macrophage counts during infection with Pneumocystis carinii and recovery.

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. carinii-infected 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.

Evidence for the requirement of T cell costimulation in the pathogenesis of natural Pneumocystis carinii pulmonary infection.

Pneumocystis carinii pneumonia (PCP) is a frequent and serious opportunistic infection in immunocompromized patients. Although the pathogenesis of PCP-mediated lung injury is poorly understood, a central involvement of host inflammatory responses has been implicated. We have found that while the loss of specific T cell costimulatory signals increases susceptibility to the spontaneous pneumocystis infection, PCP-induced pulmonary injury (and subsequent morbidity and mortality) involves other intact costimulatory pathways. Mice that are genetically deficient for the costimulatory receptor CD154 (CD154 knockout (ko) mice) spontaneously developed PCP, consistent with the increased susceptibility of X-linked hyper IgM syndrome patients (caused by CD154 gene mutations) to P. carinii infection. In these mice PCP was manifested by progressive weight loss, dyspnea and death. In contrast, CD154 ko mice also genetically lacking ICAM1 (CD154 koxICAM1 ko) or CD28 (CD154 koxCD28 ko) costimulatory receptors had later onset of weight loss and significantly prolonged survival. Although onset of infection and age-matched P. carinii organism burden were equivalent, the CD154 single knockout mice had evidence of greater pulmonary inflammation vs. the double ko's. These findings suggest that costimulation-dependent T cell-mediated inflammation plays an important role in both susceptibility to and pathogenesis of PCP, and may identify potential molecular targets for novel immunomodulatory treatment approaches.