Pneumocystis carinii attachment increases expression of fibronectin-binding integrins on cultured lung cells.

Pneumocystis carinii is an extracellular pathogen which requires attachment to alveolar epithelial cells for growth and replication. Previous studies have demonstrated that the extracellular matrix protein fibronectin (Fn) facilitates attachment of P. carinii to lung cells. This study addresses the role of cell surface Fn receptors (integrins) as mediators of P. carinii attachment and demonstrates the effect of P. carinii attachment on integrin expression on cultured lung cells. To determine the role of Fn-binding integrins in P. carinii attachment, attachment of 51Cr-labelled P. carinii organisms to the lung epithelial cell line A549 was quantified in the presence or absence of anti-integrin antibodies. Antibodies to the alpha v and alpha 5 integrin subunits significantly inhibited P. carinii attachment, while addition of antibody to the alpha subunit of a non-Fn-binding integrin, alpha 2, did not affect P. carinii attachment. To further investigate the role of Fn-binding integrins in P. carinii attachment, the effect of P. carinii attachment on expression of the alpha v and alpha 5 integrin subunits was determined. A549 cells incubated with either P. carinii organisms or with the P. carinii major surface glycoprotein gp120 demonstrated a 3- to 10-fold increase in expression of the alpha 5 integrin subunit; however, neither P. carinii nor gp120 affected the expression of alpha v integrin. Furthermore, the effects of P. carinii on A549 cell alpha 5 integrin expression were attenuated by the addition of an anti-gp120 antibody which blocks P. carinii attachment to A549 cells. Therefore, P. carinii attachment to lung epithelial cells appears to be mediated by alpha v- and alpha 5-containing integrins expressed on the epithelial cell surface, and P. carinii attachment results in increased expression of the alpha 5 integrin subunit.     

Lung epithelial cells and extracellular matrix components induce expression of Pneumocystis carinii STE20, a gene complementing the mating and pseudohyphal growth defects of STE20 mutant yeast

Pneumocystis carinii causes severe pneumonia in immunocompromised hosts. The binding of P. carinii to alveolar epithelial cells and extracellular matrix constituents such as fibronectin and vitronectin is a central feature of infection, which initiates proliferation of the organism. Herein, we demonstrate that P. carinii binding to lung cells specifically alters the gene expression of the organism, regulating fungal growth. Subtractive hybridization was performed to isolate P. carinii genes expressed following binding to mammalian extracellular matrix constituents. P. carinii STE20 (PCSTE20), a gene participating in mating and pseudohyphal growth of other fungi, was identified following adherence to the extracellular matrix constituents fibronectin, vitronectin, collagen, and lung epithelial cells. The expression of PCSTE20 and a related P. carinii mitogen-activated protein kinase (MAPK) kinase gene, also implicated in signaling of mating, were both specifically upregulated by binding to matrix protein. The expression of general cyclin-dependent kinases and other MAPKs not involved in mating pathways were not altered by organism binding. PCSTE20 expression was also strongly enhanced following organism attachment to A549 lung epithelial cells. When expressed in a Saccharomyces cerevisiae ste20Delta mutant, PCSTE20 suppressed defects in both mating and pseudohyphal growth. These findings are consistent with the observed proliferation and filopodial extension of Pneumocystis organisms adherent to the epithelium in the lungs of immunocompromised hosts. PCSTE20 expression appears to represent a significant component in the regulation of the life cycle of this intractable opportunistic pathogen.     

Carbohydrate recognition domain of surfactant protein D mediates interactions with Pneumocystis carinii glycoprotein A

Pneumocystis carinii continues to cause severe pneumonia in immunocompromised patients. Surfactant protein D (SP-D), a lung collectin, markedly accumulates during P. carinii pneumonia and binds to glycoprotein A (gpA) on the surface of P. carinii, thereby enhancing interactions with alveolar macrophages. Herein, we report the structural basis of the interaction of SP-D with gpA. We demonstrate that natural SP-D binds to purified gpA in the presence of 2 mM calcium in a saturable, concentration-dependent manner, which is abolished by 10 mM ethylenediaminetetraacetic acid. Increasing concentrations of calcium under otherwise cation-free conditions significantly enhance SP-D binding to gpA, whereas manganese and magnesium cations have minimal effect. Maximal SP-D binding occurs at pH 7.4, with significant inhibition at pH 4. SP-D binding to gpA is also competitively inhibited by maltose>glucose>mannose>N-acetyl-glucosamine. Comparison of the binding of various natural and recombinant forms of SP-D to gpA reveals that the number of carbohydrate recognition domains (CRDs) in a given SP-D form determines the relative extent of binding to gpA. Maximal binding is observed with natural SP-D (dodecamers and higher order SP-D complexes) followed by recombinant dodecamers. In contrast, recombinant full-length trimers exhibit substantially less binding, which is similar to that observed with a recombinant truncated molecule consisting of the CRD and neck regions, and containing trimers of this portion of the molecule. Taken together, these findings strongly indicate that the CRD of SP-D mediates interaction with P. carinii gpA through its attached oligosaccharides and that the extent of SP-D binding to P. carinii is greatest with dodecamers and higher order forms of SP-D.     

A soluble mannose receptor immunoadhesin enhances phagocytosis of Pneumocystis carinii by human polymorphonuclear leukocytes in vitro

Pneumocystis carinii is an opportunistic pathogen that causes pneumonia in immunocompromised hosts. In the normal host, P. carinii is susceptible to an array of first line host defense mechanisms that are operative in the lung. Alveolar macrophages play a central role in the clearance of inhaled organisms. The macrophage mannose receptor (MR) appears to be sufficient for P. carinii phagocytosis. In individuals infected with the human immunodeficiency virus, MR expression on alveolar macrophages and P. carinii phagocytosis are decreased, however, Fc-receptor mediated phagocytosis remains intact. In this study, we demonstrate that a recombinant soluble MR immunoadhesin, consisting of the essential carbohydrate binding MR ectodomain and the Fc-region of human immunoglobulin (Ig)G1, binds P. carinii and leads to an 8.2-fold increased uptake of P. carinii by phagocytic cells. Our results suggest that the soluble MR immunoadhesin may have therapeutic potential in the treatment of P. carinii infections.     

Clinical and experimental studies on inflammatory mediators during AIDS-associated Pneumocystis carinii pneumonia

This thesis is based on studies carried out during my appointment as a research fellow at the Department of Infectious Diseases, Hvidovre Hospital, University of Copenhagen, Denmark from 1993 to 1997. Part of this period was spent as a guest researcher at the Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland, USA. Pneumocystis carinii pneumonia (PCP) is the most frequent AIDS defining illness over the past 20 years. PCP is associated with considerable morbidity and mortality. An inflammatory reaction to P. carinii is believed to cause respiratory failure. This thesis has attempted to delineate important mechanisms of the inflammatory cascade, and to determine how inflammation is initiated during PCP. In histopathological studies of lung specimens it was shown that PCP caused significant inflammation and destruction of tissue. Specific pathological changes of the alveolar epithelium was observed in PCP but not for other HIV related lung diseases. By determining concentrations of soluble markers of immune activation we found that anti-microbial therapy exacerbated an ongoing inflammatory reaction. Adjuvant glucocorticosteroids suppressed levels of soluble immune markers. Bronchoalveolar lavage (BAL) neutrophilia has been associated with disease severity, and an increased risk of death from PCP. Through competitive inhibitory studies, we showed that BAL fluid neutrophil chemotactic activity largely was explained by the presence of interleukin-8 (IL-8). Further, we showed a correlation between high levels of BAL fluid IL-8 and mortality. Adjuvant treatment with glucocorticosteroids lowered BAL fluid IL-8 levels. In experimental studies we found that P. carinii Major Surface Antigen (MSG) induced IL-8 and tumor necrosis factor-alpha secretion from human monocytes and an alveolar epithelial cell line (A549). Binding of MSG to monocytes appeared to be mediated by mannose receptors, while A549 cells recognized MSG through mannose and glucan receptors. Glucocorticosteroids attenuated IL-8 secretion from A549 cells. These studies have confirmed that P. carinii infection induces tissue damage through a significant inflammatory response initiated by secretion of inflammatory mediators. Glucocorticosteroids attenuates the inflammatory response.     

Pneumocystis carinii glycoprotein A binds macrophage mannose receptors.

Pneumocystis carinii causes life-threatening pneumonia in patients with impaired immunity. Recent studies suggest that alveolar macrophages interact with P. carinii through macrophage mannose receptors. However, the ligand(s) on P. carinii that is recognized by these receptors has not been fully defined. P. carinii contains a major mannose-rich surface antigen complex termed glycoprotein A (gpA). It was therefore hypothesized that gpA binds directly to macrophage mannose receptors and mediates organism attachment to these phagocytes. To assess this, gpA was purified from P. carinii by continuous-elution gel electrophoresis. 125I-labeled gpA bound to alveolar macrophages in a saturable fashion. In addition, gpA binding was substantially inhibited by both alpha-mannan and EDTA, further suggesting that gpA interacts with macrophage mannose receptors. Macrophage membrane proteins capable of binding to gpA were isolated with a gpA-Sepharose column. A 165-kDa membrane-associated protein was specifically eluted from the gpA-Sepharose column with EDTA (20 mM). This protein was identified as the macrophage mannose receptor by immunoprecipitation with a polyclonal anti-mannose receptor antiserum. To further investigate the role of gpA in P. carinii-macrophage interactions, 51Cr-labeled P. carinii cells were incubated with macrophages in the presence of increasing concentrations of soluble gpA, and organism attachment was quantified. Soluble gpA (2.5 mg/dl) competitively inhibited P. carinii attachment to alveolar macrophages by 51.3% +/- 3.7% (P = 0.01). Our findings demonstrate that gpA present on P. carinii interacts directly with mannose receptors, thereby mediating organism attachment to alveolar macrophages.     

Gamma interferon stimulates rat alveolar macrophages to kill Pneumocystis carinii by L-arginine- and tumor necrosis factor-dependent mechanisms

Pneumocystis carinii pneumonia remains a serious complication for immunocompromised patients. In the present study, P. carinii organisms interacted with gamma interferon (IFN-gamma)-stimulated alveolar macrophages (AMs) to activate the L-arginine-dependent cytocidal pathway involving reactive nitrogen intermediates (RNI) that were assayed as nitrite (NO2-). Unstimulated cultures of AMs produced negligible quantities of RNI. Addition of P. carinii organisms to IFN-gamma-primed AMs resulted in greatly enhanced production of RNI. NO2- levels increased from 0.8 +/- 0.4 to 11.1 +/- 3.8 microM as early as 6 h after P. carinii organisms were incubated with IFN-gamma-stimulated AMs and to 35.1 +/- 8.9 microM after a 24-h incubation, a near-maximum level. High levels of NO2- were produced by AMs primed with as little as 10 U of IFN-gamma per ml in the presence of P. carinii, and a 20-fold increase in IFN-gamma concentration resulted in only a further 65% increase in NO2- production. RNI-dependent killing of P. carinii was demonstrated by both a 51Cr release assay and a [35S]methionine pulse immunoprecipitation assay. Addition of either monoclonal tumor necrosis factor alpha (TNF-alpha) neutralizing antibody or 200 microM NG-monomethyl-L-arginine (L-NGMMA), a competitive inhibitor of the L-arginine-dependent pathway, significantly decreased NO2- production and reduced P. carinii killing. TNF-alpha alone had no effect on P. carinii viability. These results suggest that (i) the specific interaction of P. carinii organisms with IFN-gamma-primed AMs triggers the production of RNI, (ii) RNI are toxic to P. carinii, and (iii) TNF-alpha likely plays a central role in mediating P. carinii killing by IFN-gamma-stimulated AMs.     

Alveolar response to experimental Pneumocystis carinii pneumonia in the rat.

In order to characterize the alveolar response to Pneumocystis carinii pneumonia, light and electron miscropy were used to trace the development of experimental infections with P carinii in rats treated with cortisone acetate and a low-protein diet. The first changes were found by the eighth day of treatment and consisted of the selective attachment of Pneumocystis organisms, mostly trophozoites, to alveolar Type 1 pneumocytes; the host cells were undamaged, and no inflammatory response was seen. After approximately one month of treatment, the seemingly innocuous host-parasite interaction was succeeded by focal necrosis of the Type 1 pneumocytes adjacent to organisms; hyperplasia of nearby Type 2 pneumocytes also occurred, to replace the dead Type 1 pneumocytes. Even at this stage, inflammatory reaction was conspicuously absent except for occasional alveolar macrophages in the diseased alveoli; in addition, all cells of the alveolar-capillary membrane other than Type 1 pneumocytes appeared entirely normal. Not only does the present study clarify the nature of alveolar injury caused by Pneumocystis carinii, but it also provides an experimental animal model in which selective injury of the alveolar lining cells occurs.     

Granulomatous Pneumocystis carinii pneumonia: DNA amplification studies on bronchoscopic alveolar lavage samples.

Three HIV positive subjects presented with symptoms and radiographic changes suggestive of Pneumocystis carinii pneumonia. Methenamine silver staining of bronchoscopic alveolar lavage (BAL) fluid was negative (from one sample in one patient and two samples in the other two patients). Open lung biopsy was performed because of uncertain clinical progress and diagnosis; all three patients were found to have multiple pulmonary granulomata encasing numerous P carinii organisms. DNA amplification, using P carinii specific oligonucleotides, was performed on stored bronchoscopic BAL samples. P carinii specific amplification product was detected by ethidium bromide staining after electrophoretic separation on agarose gel in one case, and by the more sensitive technique of oligohybridisation in all three cases. In granulomatous P carinii pneumonia organisms are rarely identified in bronchoscopic alveolar lavage samples using histochemical staining, but are detectable by DNA amplification, although not at levels which can be readily distinguished from low, subclinical infection.     

Toxicity of pneumolysin to pulmonary alveolar epithelial cells.

Mortality during the first several days of pneumococcal pneumonia has not decreased appreciably over the past 30 years, despite the widespread use of antibiotics. Disruption of the alveolar epithelial barrier is likely an initial step in the pathogenesis of pneumococcal pneumonia. We report that soluble factors from Streptococcus pneumoniae can directly injure isolated rat alveolar epithelial cells. Using biochemical and immunological techniques, we identified pneumolysin as a major soluble S. pneumoniae toxin for alveolar epithelial cells. Alveolar epithelial cells at 24 or 72 h after isolation were equally sensitive to injury by purified pneumolysin. Purified pneumolysin substantially increased alveolar permeability in an isolated perfused rat lung model. Electron microscopy revealed that instilled pneumolysin caused widespread lung injury, primarily to type I alveolar epithelial cells. Pneumolysin toxicity to alveolar epithelial cells may be important in the pathogenesis of acute lung injury during pneumococcal pneumonia and may facilitate pneumococcal bacteremia.     

Reconstitution of immune responses to Pneumocystis carinii pneumonia in patients with HIV infection who receive highly active antiretroviral therapy

We describe a reconstitution syndrome of immune responses to Pneumocystis carinii pneumonia (PCP) in 2 HIV-infected individuals who received highly active antiretroviral therapy (HAART). Patient 1, who had been successfully treated for PCP 3 years before the initiation of HAART, developed cough and pulmonary shadows 6 weeks after the start of HAART. Patient 2 was introduced HAART immediately after completing the responsive treatment of PCP, and then showed dyspnea and diffuse pulmonary infiltrates 7 months later. Histologic findings of lung-tissue samples showed granulomatous tissue (patient 1) and organizing pneumonia with thickening of alveolar septa (patient 2), and immunohistochemical findings revealed both CD4 and CD8 cell subsets represented in the lesions. The tissue and bronchoalveolar lavage (BAL) specimens showed no organisms, but PCR methods with the BAL samples were positive for P. carinii DNA. It is hypothesized that these second respiratory episodes may have arisen as immune reconstitution syndrome in response to residual P. carinii antigen in the lung.     

Phospholipid profile of Pneumocystis carinii and its interaction with alveolar type II epithelial cells.

Pneumocystis carinii is an obligate parasite of mammalian lungs, attaching to but not invading the alveolar epithelium. The alveolar air spaces are rich in phospholipids, which are secreted by steroid-responsive alveolar type II epithelial cells. P. carinii isolated from rat lungs was found to contain the expected structural phospholipids as well as a large amount of firmly attached disaturated phosphatidylcholine, the characteristic phospholipid of alveolar surfactant. In vitro, P. carinii cells synthesized phospholipids from simple radiolabeled precursors; disaturated phosphatidylcholine was not formed. However, washed P. carinii cells avidly adsorbed radiolabeled rat surfactant, a process that appeared to be saturable, not dependent on viability of the organisms, and abolished by incubation at 4 degrees C. The surfactant was neither harmful nor beneficial to in vitro survival of the organisms. With the exception of high concentrations of arachidonic acid, fatty acids found in rat alveolar lining material were also not toxic. In addition, cultures consisting primarily of rat type II alveolar epithelial cells were toxic to P. carinii when the organisms were added to monolayers of type II cells at less than or equal to 10:1 multiplicity. At higher multiplicities, the parasite survived (but did not increase in numbers), and the type II cells deteriorated. The mechanism for this effect has not been determined.     

Pseudomonas aeruginosa outer membrane protein F is an adhesin in bacterial binding to lung epithelial cells in culture

Adherence to host cells is a crucial step by which bacteria initiate an infection but the bacterial determinants of the process are, as yet, poorly understood. In an effort to identify bacterial adhesins responsible for Pseudomonas aeruginosa binding to host cells, we identified porin F (OprF) from the outer membrane of P. aeruginosa as adhesin for human alveolar epithelial (A549) cells. Bacterial adhesion assays with (35)S-labeled wild type P. aeruginosa and its isogenic mutant strain lacking OprF showed that the mutant strain binds 43% less than the wild type to A549 cells (P<0.01). In addition, bacterial binding is significantly reduced (P<0.01) when either A549 cells were pretreated with purified OprF or if bacteria were pre-incubated with a monoclonal antibody to OprF. Finally, ligand binding experiments in which purified OprF protein was added to A549 monolayers showed saturable binding. These data indicate that OprF contributes to bacterial adherence to A549 epithelial cells and could facilitate Pseudomonas interactions with the epithelium, including colonization of the airway epithelium or the initiation of pulmonary infection.     

Surfactant protein D-mediated aggregation of Pneumocystis carinii impairs phagocytosis by alveolar macrophages

Pneumocystis carinii remains an important and potentially fatal cause of opportunistic pneumonia. Animal studies reveal that substantial quantities of surfactant protein D (SP-D) accumulate in the airspaces during P. carinii pneumonia and are particularly abundant in aggregates of organisms. Due to the multimeric structure of SP-D, we hypothesized that SP-D mediates aggregation of the organism. From previous clinical studies it is known that aggregated organisms are conspicuous in sections of lung tissue and bronchoalveolar lavage (BAL) fluids of humans with active P. carinii pneumonia. Herein, we observe that SP-D levels increased at least fourfold in BAL fluids of patients with P. carinii pneumonia. Next, a spectrophotometric sedimentation assay was developed to assess the aggregation of P. carinii in vitro by SP-D. P. carinii organisms were first stripped with glutathione to remove bound SP-D and subsequently incubated in the presence of SP-D and 2 mM calcium. P. carinii incubated with natural SP-D (10 micro g/ml) containing dodecamers and higher-order forms exhibited aggregation and enhanced sedimentation compared to that of glutathione-stripped P. carinii. Aggregation was also enhanced by the concentrated supernatant of rat BAL fluid, and this effect was abolished by the selective removal of SP-D from the lavage fluid. P. carinii aggregation was reduced by maltose, mannose, and EDTA, consistent with the role of the SP-D C-type lectin domain (CRD) in the aggregation event. Comparisons of different molecular forms of SP-D showed that dodecamers-but not trimeric subunits-mediate optimal aggregation of P. carinii. Aggregation of P. carinii by SP-D was shown to be responsible for the impaired phagocytosis of the organisms by alveolar macrophages. Thus, SP-D-mediated aggregation of P. carinii may represent one means by which the organism avoids elimination by the host.     

Growth and serial passage of Pneumocystis carinii in the A549 cell line

Pneumocystis carinii obtained from infected rats and patients was cultured in the A549 cell line, a presumptive alveolar type 2 cell line derived from a human lung carcinoma. Standard criteria were established for organism sampling, quantitation, and growth. The trophozoite form of P. carinii was a more sensitive indicator of growth than was the cyst. Rat P. carinii increased 10-fold in primary culture and could be serially passed three additional times to new cultures; success in growing human P. carinii was limited and appeared to be related to the quality of the specimen received for culture. Growth pattern experiments suggested that close interaction of P. carinii with the cell monolayer is an important step in the life cycle of the organism. Thus, the A549 culture system should be useful for in vitro studies of the immunobiology of P. carinii.     

Lung mechanics,radiography and 67Ga scintigraphy in experimental Pneumocystis carinii pneumonia.

Respiratory system pressure-volume (P-V) studies, 67Ga-citrate scans, and chest radiographs were made in the corticosteroid-treated rat model of Pneumocystis carinii pneumonia. The steroid treatment used to provoke Pneumocystis infection in this model resulted in a reduction in body weight, lung weight and lung volumes compared to age-matched controls but no change in the normalized pressure-volume curve. P. carinii infection was associated with increased lung wet weight and flattening of the respiratory system P-V curve when compared to either age matched controls or steroid-treated animals on trimethoprim-sulfamethoxazole prophylaxis for P. carinii. Radiographs were interpreted as positive in only three of 11 animals with P. carinii, whereas 10 of 12 animals showed positive gallium-67 lung scans. We conclude that both gallium uptake and altered lung mechanics occur in the rat with Pneumocystis carinii and may reflect increased alveolar permeability and surfactant abnormalities noted in other studies.     

Lung mechanics, radiography and 67Ga scintigraphy in experimental Pneumocystis carinii pneumonia

Respiratory system pressure-volume (P-V) studies, 67Ga-citrate scans, and chest radiographs were made in the corticosteroid-treated rat model of Pneumocystis carinii pneumonia. The steroid treatment used to provoke Pneumocystis infection in this model resulted in a reduction in body weight, lung weight and lung volumes compared to age-matched controls but no change in the normalized pressure-volume curve. P. carinii infection was associated with increased lung wet weight and flattening of the respiratory system P-V curve when compared to either age matched controls or steroid-treated animals on trimethoprim-sulfamethoxazole prophylaxis for P. carinii. Radiographs were interpreted as positive in only three of 11 animals with P. carinii, whereas 10 of 12 animals showed positive gallium-67 lung scans. We conclude that both gallium uptake and altered lung mechanics occur in the rat with Pneumocystis carinii and may reflect increased alveolar permeability and surfactant abnormalities noted in other studies.     

Pneumocystis carinii induction of tumor necrosis factor-alpha by alveolar macrophages: modulation by pentamidine isethionate.

Pneumocystis carinii, and the inflammatory response it provokes, together contribute to irreversible lung damage in immunocompromised patients. P. carinii cysts were found to be capable of inducing tumor necrosis factor-alpha (TNF) release from alveolar macrophages in a concentration-dependent manner. At physiologically achievable concentrations, pentamidine isethionate (pentamidine) substantially reduces such production. Pretreatment of alveolar macrophages (AM phi) with interferon-gamma (IFN-gamma) synergizes with P. carinii to produce increased levels of TNF, a condition which pentamidine was also able to antagonize. Pentamidine treatment did not interfere with the phagocytic ability of AM phi. Considering clinical reduction of TNF could lessen P. carinii pneumonia (PCP) induced inflammation, the efficacy of pentamidine in the treatment of PCP may be partially associated with its ability to inhibit the release of inflammatory mediators such as TNF.