Steve D. Swain

Alveolar Macrophage–mediated Killing of Pneumocystis carinii f. sp. muris Involves Molecular Recognition by the Dectin-1 β-Glucan Receptor

Innate immune mechanisms against Pneumocystis carinii, a frequent cause of pneumonia in immunocompromised individuals, are not well understood. Using both real time polymerase chain reaction as a measure of organism viability and fluorescent deconvolution microscopy, we show that nonopsonic phagocytosis of P. carinii by alveolar macrophages is mediated by the Dectin-1 β-glucan receptor and that the subsequent generation of hydrogen peroxide is involved in alveolar macrophage–mediated killing of P. carinii. The macrophage Dectin-1 β-glucan receptor colocalized with the P. carinii cyst wall. However, blockage of Dectin-1 with high concentrations of anti–Dectin-1 antibody inhibited binding and concomitant killing of P. carinii by alveolar macrophages. Furthermore, RAW 264.7 macrophages overexpressing Dectin-1 bound P. carinii at a higher level than control RAW cells. In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcγRII/III receptors. Opsonized P. carinii could also be efficiently killed in the presence of FcγRII/III receptor blockage through Dectin-1–mediated phagocytosis. We further show that Dectin-1 is required for P. carinii–induced macrophage inflammatory protein 2 production by alveolar macrophages. Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 β-glucan receptor.

Neutrophil Priming in Host Defense: Role of Oxidants as Priming Agents

Neutrophils play an essential role in the bodys innate immune response to infection. To protect the host, these phagocytic cells possess an impressive array of microbicidal weapons that can be brought to bear on an invading pathogen, including a variety of toxic oxygen radical species and proteolytic enzymes. Although the neutrophil response is designed to restrict the damage to the smallest possible region where the pathogen is located, some of the damaging agents inevitably leak into the surrounding areas where they have the capacity to inflict tissue damage at sites of inflammation. Thus, it is essential that the host defense response of these cells is finely tuned to result in the appropriate level of response to any given situation. One of the regulatory mechanisms implicated in controlling neutrophil responses is priming. Through the action of priming agents, the level of activation and subsequent responses of the cell can be regulated so that a continuum of activation states is achieved. In this review, we describe key features of the priming response in host defense and disease pathogenesis and focus on the unique role of reactive oxygen species as priming agents.

Absence of the Macrophage Mannose Receptor in Mice Does Not Increase Susceptibility to Pneumocystis carinii Infection In Vivo

ABSTRACT Host defense against the opportunistic pathogen Pneumocystis carinii requires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance of P. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis of P. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor in P. carinii clearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection by P. carinii in otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible to P. carinii by CD4+ depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated during P. carinii infection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition of P. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.

ANALYSIS OF ACTIVATION-INDUCED CONFORMATIONAL CHANGES IN P47PHOX USING TRYPTOPHAN FLUORESCENCE SPECTROSCOPY

Activation of the neutrophil NADPH oxidase requires translocation of cytosolic proteins p47 phox , p67 phox , and Rac to the plasma membrane or phagosomal membrane, where they assemble with membrane-bound flavocytochrome b. During this process, it appears that p47 phox undergoes conformational changes, resulting in the exposure of binding sites involved in assembly and activation of the oxidase. In the present study, we have directly evaluated activation-induced conformational changes in p47 phox using tryptophan fluorescence and circular dichroism spectroscopy. Treatment of p47 phox with amphiphilic agents known to activate the NADPH oxidase (SDS and arachidonic acid) caused a dose-dependent quenching in the intrinsic tryptophan fluorescence of p47 phox , whereas treatment with a number of other amphiphilic agents that failed to activate the oxidase had no effect on p47 phox fluorescence. In addition, the concentration range of activating agents required to induce changes in fluorescence correlated with the concentration range of these agents that induced maximal NADPH oxidase activity in a cell-free assay system. We next determined if activation by phosphorylation caused the same type of conformational changes in p47 phox . Protein kinase C phosphorylation of p47 phox in vitro resulted in comparable quenching of fluorescence, which also correlated directly with NADPH oxidase activity. Finally, the circular dichroism (CD) spectrum of p47 phox was significantly changed by the addition of SDS, whereas treatment with a non-activating detergent had no effect on the CD spectrum. These results support the conclusion that activation by amphiphilic agents results in changes in the secondary structure of p47 phox . Thus, our studies provide direct evidence linking conformational changes in p47 phox to the NADPH oxidase activation/assembly process and also further support the hypothesis that amphiphile-mediated activation of the NADPH oxidase induces changes in p47 phox that are similar to those mediated by phosphorylation in vivo.

Inducible Bronchus-Associated Lymphoid Tissue Elicited by a Protein Cage Nanoparticle Enhances Protection in Mice against Diverse Respiratory Viruses

Background Destruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage. Methodology/Principal Findings Mice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms. Conclusions/Significance The immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.

Neither neutrophils nor reactive oxygen species contribute to tissue damage during Pneumocystis pneumonia in mice.

ABSTRACT Neutrophils are implicated in the damage of lung tissue in many disease states, including infectious diseases and environmental insults. These effects may be due to oxidative or nonoxidative functions of the neutrophil or both. We examined the role of neutrophils in pulmonary damage during infection with the opportunistic fungal pathogen Pneumocystis sp. in four mouse models of neutrophil dysfunction. These were (i) a knockout of the gp91phox component of NADPH oxidase, in which reactive oxygen species (ROS) production is greatly reduced; (ii) a double knockout of gp91phox and inducible nitric oxide synthase, in which ROS and nitric oxide production is greatly decreased; (iii) a knockout of the chemokine receptor CXCR2, in which accumulation of intra-alveolar neutrophils is severely diminished; and (iv) antibody depletion of circulating neutrophils in wild-type mice with the monoclonal antibody RB6. Surprisingly, in each case, indicators of pulmonary damage (respiratory rates, arterial oxygen partial pressures, and intra-alveolar albumin concentrations) were the same in knockout mice and comparable wild-type mice. Therefore, whereas neutrophils are a valid correlative marker of lung damage during Pneumocystis infection, neither neutrophils nor ROS appear to be the causative agent of tissue damage. We also show that there is no difference in Pneumocystis burdens between wild-type and knockout mice, which supports the idea that neutrophils do not have a major role in the clearance of this organism.

Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite‐mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN‐1) modified actin in a concentration‐dependent manner, resulting in an inhibition of globular‐actin polymerization and filamentous‐actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU‐101033E, which has been shown previously to specifically block peroxynitrite‐mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite‐treated actin demonstrated a concentration‐dependent increase in nitrotyrosine, which was also blocked by PNU‐101033E. Activation of neutrophils in the presence of a nitric oxide donor (S‐nitroso‐N‐acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite‐treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin‐dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN‐1 treatment in a concentration‐dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin‐dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Priming of human neutrophils by peroxynitrite: potential role in enhancement of the local inflammatory response.

Peroxynitrite is a potent oxidant generated from the reaction of nitric oxide (NO) and superoxide anion (O2−), both of which can be produced in inflammatory tissues. In these studies, we analyzed what direct effect peroxynitrite had on neutrophil (PMN) function. We found that peroxynitrite was an effective priming agent for PMNs, as demonstrated by enhanced O2− production on subsequent activation with low doses of PMA or N‐formyl‐methionine‐leucine‐phenylalanine (fMLF), changes in the expression of PMN surface markers (L‐selectin, Mac‐1, flavocytochrome b, and fMLF receptor), and increased intracellular calcium levels. Analysis of the mechanism of PMN priming by peroxynitrite demonstrated that peroxynitrite resulted in minimal oxidation of protein sulfhydryl groups and subsequent protein cross‐linking. In contrast, treatment of PMNs with peroxynitrite resulted in significant nitration of tyrosine residues on neutrophil proteins. In addition, inhibition of tyrosine nitration with a pyrrolopyrimidine antioxidant blocked the majority of peroxynitrite‐induced priming effects, further suggesting that PMN priming was mediated primarily by nitration of tyrosine residues on PMN proteins. The ability of peroxynitrite to serve as an effective priming agent for PMNs at sites of inflammation may play a key role in modulating the host‐defense process. J. Leukoc. Biol. 65: 59–70; 1999.

Role of Type I IFNs in Pulmonary Complications of Pneumocystis murina Infection

Despite the advent of highly active antiretroviral therapy, pulmonary complications in AIDS are a common clinical problem. Pneumocystis jiroveci infection causes a life-threatening pneumonia, especially in individuals with CD4 T cell deficiencies as occurs in AIDS. Although Pneumocystis sp. is an extracellular fungal pathogen, CD8 T cells are the predominant lymphocyte recruited to the lung in CD4-deficient humans and mice during Pneumocystis pneumonia, and we have found that these CD8 T cells are responsible for subsequent lung damage in CD4 T cell-depleted mice. Comparing CD4 T cell-depleted IFN-α receptor knockout (KO) mice to wild-type mice, we found that this CD8 T cell recruitment and lung damage is type I IFN (IFN-αβ) dependent. However, in both CD4 competent, wild-type and IFN-α receptor (IFNAR) KO mice, Pneumocystis infection leads to an eosinophilic granulocyte influx with bronchial epithelial changes as seen in asthma. This response is delayed in IFNAR KO mice, as is pathogen clearance. Although the inflammation is transient in wild-type animals and resolves upon Pneumocystis clearance, it is more severe and persists through day 35 postinfection in IFNAR KO mice, leading to fibrosis. In addition, IFNAR KO, but not wild-type, mice mount a Pneumocystis-specific IgE response, an indicator of allergic sensitization. Thus, in the absence of IFNAR signaling and CD4 T cells, Pneumocystis-mediated lung damage does not occur, whereas in CD4-competent animals, the absence of IFNAR signaling results in an exacerbated Th2 response, asthma-like symptoms, and fibrosis. Therefore, both CD4 T cell- and type I IFN-mediated mechanisms can determine pulmonary complications from Pneumocystis infection.

Platelet-activating factor induces a concentration-dependent spectrum of functional responses in bovine neutrophils.

We characterized the dose response of bovine neutrophils to platelet‐activating factor (PAF) with respect to the following functions: calcium flux and membrane potential changes, actin polymerization, degranulation, and the production and/or priming of the oxidative burst. PAF at very low concentrations (10 and 10 M) caused changes in intracellular calcium and membrane potential in bovine neutrophils, whereas moderate PAF concentrations (≥10–7 M) resulted in increased actin polymerization. Degranulation responses to PAF were more complex: low concentrations (10–9 M) caused secretory granule degranulation, moderate doses (≥ 10–7 M) caused specific granule degranulation, whereas azurophil degranulation only occurred at high (10–5 M) PAF concentrations. Treatment of bovine neutrophils with PAF at concentrations ≥10–7 M also caused up‐regulation of the adhesion molecules Mac‐1 and L‐selectin. PAF stimulation resulted in a very weak [compared to phorbol myristate acetate (PMA)] oxidative burst in bovine neutrophils, and only at high (10–6 M) concentrations. Unlike human neutrophils, bovine neutrophils were poorly primed by PAF treatment. Only high concentrations of PAF (10–5 M) caused an increased rate of PMA‐stimulated superoxide production, although lower doses of PAF did reduce the lag time preceding the PMA‐induced oxidative burst. The overall pattern that can be inferred is that lower concentrations of PAF promote neutrophil sensitivity and interaction by selective degranulation, up‐regulation of adhesion molecules, and increased actin polymerization. In contrast, higher PAF concentrations can promote, albeit weakly, more direct bactericidal responses, such as the release of reactive oxygen species and granule enzymes. The ability of PAF to modulate a graded response in bovine neutrophils would allow the cell to respond proportionally to the severity of a stimulus. J. Leukoc. Biol. 64: 817–827; 1998.