Proliferative and cytokine responses to a major surface glycoprotein of Pneumocystis carinii.

Naturally derived T-cell responses by rats to a 120-kDa major surface glycoprotein (MSG) of rat-derived Pneumocystis carinii were analyzed in vitro. Specific cytokines elicited by the T-cell response to the MSG were also identified. MSG was purified from rat-derived P. carinii by three different techniques: lectin affinity chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution, and size-exclusion high-performance liquid chromatography. The cell-mediated immunity of spleen cells isolated from Lewis rats with and without natural exposure to P. carinii to the purified MSG was studied. Exposure to P. carinii was monitored by the presence or absence of serum antibodies to P. carinii antigens by Western blotting (immunoblotting). A T-cell proliferative response to the MSG was identified only with spleen cells isolated from rats exposed to P. carinii and peaked at 4 days. Flow cytometric analysis revealed that the percentage of CD4 cells was significantly increased during the proliferative response to MSG. MSG also elicited secretion of tumor necrosis factor alpha, interleukin-1, and interleukin-2, with peak activity of these cytokines occurring after 12, 24, and 48 h, respectively, of culture. These findings suggest that MSG is important in host T-cell recognition of and immune response to P. carinii by recruitment of inflammatory cells and cytokine production.     

Enhanced lung clearance of antigen in immunised mice

A mouse model is described which enables a semi-quantitative assessment of antigen clearance from the lung. The distribution of antigen presented to the non-sensitised lung is examined and shown to be reproducible using a simple in vivo inoculation technique. Preliminary studies demonstrate that antigen clearance is significantly enhanced after parenteral immunisation and that the effect is antigen specific. Applications of the model to further studies of respiratory challenge are suggested.     

Cellular and humoral immune responses of mice subclinically infected with Pneumocystis carinii.

Cellular and humoral immune responses to Pneumocystis carinii were investigated. ICR and DDD mice were intranasally infected with 10(4) mouse lung-derived P. carinii, and the delayed-type hypersensitivity (DTH) reaction and antibody titers to P. carinii were measured along with the number of P. carinii cysts in the lungs after infection. The number of P. carinii cysts in the lungs peaked at 2 weeks after infection and then decreased to barely detectable levels by 4 weeks. Serum antibody (immunoglobulin G) titers measured by indirect immunofluorescence increased up to 4 weeks. The DTH footpad reaction was most prominent at 2 weeks postinfection and declined thereafter. Thus, the decline in the number of P. carinii cysts in the lungs corresponded well with the time of the peak of the DTH reaction but not with the serum antibody response. Spleen T cells from infected mice mediated the DTH reaction when transferred intravenously into normal recipients and reduced the number of P. carinii cysts in the lungs when transferred intravenously into P. carinii-infected mice. The results indicated that cellular immunity is important for protection from subclinical P. carinii infections.     

Inflammatory responses to Pneumocystis carinii in mice selectively depleted of helper T lymphocytes.

Pneumocystis carinii is the most important pulmonary pathogen in patients with the acquired immunodeficiency syndrome, but host defenses against P. carinii are not well characterized. We recently reported an experimental model of P. carinii infection, in which mice selectively depleted of CD4+ lymphocytes develop pulmonary infection after inoculation with P. carinii. In the current study, we compared lung inflammatory responses to P. carinii inoculation in CD4-depleted mice and in normal mice in order to further characterize host defenses against P. carinii. We hypothesized that depletion of CD4+ lymphocytes would prevent recruitment and activation of inflammatory cells in the lungs of these mice, allowing progressive infection with P. carinii. We found that CD4-depleted mice were unable to recruit CD4+ lymphocytes into their lungs and developed progressive infection with P. carinii, but mounted exuberant inflammatory responses to the organisms. These inflammatory responses were characterized by perivascular infiltration with mononuclear cells, increases in cell numbers in bronchoalveolar lavage (particularly CD8+ lymphocytes), and activation of alveolar macrophages (enhanced Ia antigen expression). In contrast, normal mice recruited CD4+ lymphocytes into their lungs and eliminated organisms with only minimal inflammatory responses. We conclude that depletion of CD4+ lymphocytes does not prevent the recruitment and activation of inflammatory cells in the lung. These inflammatory responses occur by mechanisms independent of CD4+ lymphocytes and are insufficient to provide effective host defense against P. carinii.     

Serum antibody responses to Pneumocystis carinii among different strains of normal and athymic mice.

Pneumocystis carinii infection was produced in normal mice by the administration of corticosteroids and in athymic mice by the transmission of exogenous mouse- or rat-derived organisms. Serum antibodies to P. carinii, measured by an indirect fluorescent-antibody technique, were found in five of six strains of normal mice. Although antibody titers varied widely among the control and steroid-treated mice, they were inversely proportional to the intensity of P. carinii infection in the lungs. In sequential studies, antibody titers were low during steroid administration but rose with steroid withdrawal. Antibodies were mainly of the immunoglobulin G (IgG) class; IgM antibodies also occurred, but IgA antibodies were rare. nu/nu mice rarely produced serum antibodies to P. carinii despite the fact that one strain was sensitive and the other was resistant to exogenous infection. nu/+ mice produced specific antibodies to rat and mouse P. carinii which were primarily IgG. Thus, the host produces antibodies to P. carinii which are mainly IgG and T-cell dependent; yet, these antibodies do not appear to be important in susceptibility or resistance to P. carinii infection.     

Exogenous heat-killed Escherichia coli improves alveolar macrophage activity and reduces Pneumocystis carinii lung burden in infant mice

Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis pulmonary infections. We have previously demonstrated that there is approximately a 3-week delay in the clearance of Pneumocystis organisms from pup mouse lungs compared to that in adults. We have further shown that there is approximately a 1-week delay in alveolar macrophage activation in pups versus adult mice. Alveolar macrophages are the primary effector cells responsible for the killing and clearance of Pneumocystis, suggesting that pup alveolar macrophages may be involved in the delayed clearance of this organism. Alveolar macrophages cultured in vitro with Pneumocystis alone demonstrate little to no activation, as indicated by a lack of cytokine production. However, when cultured with lipopolysaccharide (LPS) or zymosan, cytokine production was markedly increased, suggesting that pup alveolar macrophages are specifically unresponsive to Pneumocystis organisms rather than being intrinsically unable to become activated. Furthermore, pup mice treated with aerosolized, heat-killed Escherichia coli in vivo were able to clear Pneumocystis more efficiently than were control mice. Together, these data suggest that while pup alveolar macrophages are unresponsive to P. carinii f. sp. muris organisms, they are capable of activation by heat-killed E. coli in vivo, as well as LPS and zymosan in vitro. The lack of response of pup mice to P. carinii f. sp. muris may reflect protective mechanisms specific to the developing pup lung, but ultimately it results in insufficient clearance of Pneumocystis organisms.     

Activation of the respiratory burst by Pneumocystis carinii. Efficiency of different antibody isotypes,complement, lung surfactant protein D,and mannan-binding lectin

The effect of opsonization of Pneumocystis carinii with different antibody classes, complement, mannan-binding lectin (MBL), and lung surfactant protein D (SP-D) on respiratory burst activation was studied. Antibodies were obtained by affinity chromatography, complement from a hypogammaglobulinaemic patient, and phagocytic cells from blood donors. Respiratory burst activation was measured by chemiluminescence (CL). With freshly isolated neutrophils the combination of antibodies and complement but not antibody alone, had opsonizing properties. With neutrophils cultured for 20 h, however, IgG increased the CL response. In macrophages P. carinii opsonized with IgG alone induced a CL response proportional to the antibody titre used. With IgA an effect, albeit lower, was also seen, whereas IgM alone was inefficient. The combined effect of antibodies and complement increased the response significantly for all three antibody classes, IgG and complement giving the largest response. Binding of MBL to P. carinii and Candida albicans was demonstrated; however, only the former stimulated activation of the respiratory burst. SP-D did not bind to either microorganism and had no effect on the respiratory burst. It is concluded that IgG, IgA and complement are important opsonizing factors in infections involving P. carinii. The relative importance varies with the type of phagocytic cell studied.     

Development and resolution of Pneumocystis carinii pneumonia in severe combined immunodeficient mice: a morphological study of host inflammatory responses.

The development and resolution of naturally-acquired Pneumocystis carinii pneumonia was studied in a severe combined immunodeficient (SCID) mouse model by light and electron microscopies. Initial infection was evident in 3-week-old SCID mice and started as focal alveolar colonization in the areas near terminal airways. Pronounced pulmonary inflammation occurred in animals of 10 weeks or older and the infection intensity reached a plateau in animals 12 weeks of age. At this stage of disease, the histopathological features of P. carinii infection in SCID mice were similar to those of immunodeficient man. Reconstitution of SCID mice with immunocompetent spleen cells at day 0 induced substantial pulmonary inflammation that was evident already by day 7 and most severe and extensive by day 12. The clearance of P. carinii did not begin until after day 12 and was almost completed by day 17. Alveolar macrophages in mice killed between days 12 and 15, at the time when P. carinii are being rapidly cleared, appeared active but phagocytosis of P. carinii was not commonly observed by either light or electron microscopy. These results suggest that (1) the presence of non-lymphoid inflammatory cells in SCID mice is not sufficient to control P. carinii infection; (2) the clearance of P. carinii from the lungs of reconstituted SCID mice requires local recruitment of large numbers of inflammatory cells with an active appearance; and (3) intracellular killing of P. carinii by phagocytosis does not appear to be a major mechanism in host defences against P. carinii infection in this model.     

Pulmonary clearance of 99mTc--DTPA and 99mTc-albumin in rabbits with surfactant dysfunction and lung injury.

We measured the pulmonary clearance of inhaled 99mTc-DTPA and 99mTc-albumin in rabbits with surfactant dysfunction induced by dioctyl sodium sulphosuccinate and in rabbits with lung injury induced by oleic acid. The animals were tracheotomized and mechanically ventilated. After inhalation of 99mTc-albumin in ten animals, clearance of the tracer from the lungs was monitored for 90 min. The first 30 min was a control period. Dioctyl sodium sulphosuccinate was then administered in aerosol and after another 30 min oleic acid was injected intravenously. Ten other rabbits were given 99mTc-DTPA, and clearance was externally recorded for 60 min. Five animals inhaled detergent aerosol and five animals were given oleic acid intravenously after 30 min. Airway pressures, tidal volume, and arterial blood gases were measured before and after each intervention. The half-life of 99mTc-albumin in the lung was 442 +/- 123 min during the control period, 363 +/- 52 min after detergent administration, and 134 +/- 18 min after oleic acid administration (P less than 0.05 compared to control and P less than 0.01 compared to the period after detergent). The half-life of 99mTc-DTPA was 94 +/- 16 min before and 10 +/- 0.6 min (P less than 0.01) after detergent administration and 75 +/- 12 min before and 18 +/- 1.8 min (P less than 0.01) after oleic acid administration. Gas exchange was not affected by administration of dioctyl sodium sulphosuccinate but markedly impaired after injection of oleic acid. Compliance of the respiratory system remained unaffected by detergent but decreased after injection of oleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibility to Pneumocystis carinii infection: host responses of neonatal mice from immune or naive mothers and of immune or naive adults.

Mice from either naive or immunized dams were given intranasal inoculations of Pneumocystis carinii as neonates (24 to 48 h old). Lung P. carinii burdens increased through day 13 postinoculation in all pups and declined to nearly undetectable numbers by day 23 in pups from immune mothers. However, P. carinii numbers in pups from naive mothers did not begin to decline significantly until after day 33, and P. carinii organisms were still detectable in low numbers through day 45. In contrast, the lungs of naive or immunized adult mice contained detectable numbers of P. carinii organisms only up to 9 or 3 days, respectively, after inoculation. The onset of clearance of P. carinii organisms from the lungs of neonatal mice and naive adults was coincident with infiltration of neutrophils and CD4+ CD45RBlo cells into the alveolar spaces and increased titers of P. carinii-specific antibody in sera. Immunized dams had high levels of P. carinii-specific antibody in both their sera and milk, and pups from these dams had higher titers of P. carinii-specific antibody than did pups from naive dams. These data indicate that P. carinii survives for a much longer period in neonates than in adult mice, which is the result of a delay in the onset of the immune response in neonates. Furthermore, immunized mothers contributed to an early clearance of P. carinii organisms by their offspring presumably because of the transfer of P. carinii-specific antibody. However, the passively acquired antibody did not seem to have an effect until the neonates began to mount their own responses.     

Single and combined humoral and cell-mediated immunotherapy of Pneumocystis carinii pneumonia in immunodeficient scid mice.

Homozygous mutant scid/scid (severe combined immunodeficiency) mice (referred to as scid mice) lack both specific humoral and cell-mediated immune functions and are exemplary in vivo models for analysis of host-parasite relationships. In our colony, scid mice routinely and predictably develop spontaneous Pneumocystis carinii pneumonia (PCP) with high morbidity. Previous studies have identified both T cells (specifically, CD4+ cells) and antibody as independent mechanisms of effective anti-P. carinii resistance; however, CD4+ T cells also cause an often fatal hyperinflammatory reaction. The current study has explored the optimal application of these immune components for conferring protection against P. carinii. Anti-P. carinii hyperimmune serum was highly effective at reducing the number of P. carinii organisms in early, intermediate, and advanced stages of PCP and was capable of increasing the mean life expectancy of P. carinii-infected scid mice by more than threefold if provided on a continuing basis. When a short course of hyperimmune-serum therapy was provided prior to transfer of P. carinii-sensitized normal lymphocytes, scid mice were rendered permanently free of P. carinii without the pathological sequelae of the hyperinflammatory reaction. These findings are discussed in the contexts of mechanism and clinical relevance.     

Detection of antibody formation in mice, rats and rabbits immunized with different Pneumocystis carinii antigens

Antibody formation to P. carinii of human origin was determined by an indirect immunofluorescence antibody assay (IFA) after immunization of mice, rats and rabbits with pronase-treated whole cysts and soluble antigen in order to obtain more detailed data about the production of polyclonal antibodies. Antibody titre profiles over defined periods have shown that noticeable differences between the immunoreactions to whole cysts and soluble antigen occur. The soluble antigen produced an earlier and stronger titre rise (peak titres of up to 1:2560 2 to 4 weeks after immunization). In contrast to this, whole cysts produced equally high, although retarded, antibody titre peaks only in normal mice and rats and when the dose had been doubled. Nu/nu mice failed to demonstrate any reaction to these P. carinii immunogens. Cross-reactivity of the antibodies with P. carinii antigen from rat lungs was demonstrated. Possible reasons for different immunoreactions to these antigens, the importance of proteolytic digestion for the results obtained and the potential applicability of these polyclonal antibodies to a histochemical demonstration of the parasite are discussed.     

Predisposing factors in Pneumocystis carinii pneumonia: effects of tetracycline, protein malnutrition, and corticosteroids on hosts

Components of the immunosuppressive regimen used to reactivate latent Pneumocystis carinii infection were analyzed for their effects on the growth, nutrition, and lymphoid system of hosts. Rats that were administered either tetracycline or a low-protein (8%) diet alone for 7 weeks developed few abnormalities, but animals on the combined regimen developed lower body and lymphoid organ weights, lower serum albumin levels, and fewer circulating lymphocytes. Rats that were administered corticosteroids and tetracycline experienced severe wasting, debilitation, and generalized lymphocyte depletion; the low-protein diet increased the magnitude of these changes. Alterations in the frequency of occurrence of specific lymphocyte subsets occurred only in rats given corticosteroids and consisted mainly of a greater decline in peripheral blood T helper cells than in T suppressor cells. The data suggest that long-term tetracycline administration and a low-protein diet have a variety of adverse effects on the host which enhance the immunosuppressive properties of corticosteroids.     

Naturally acquired Pneumocystis carinii pneumonia in gene disruption mutant mice: roles of distinct T-cell populations in infection.

When kept under strict specific-pathogen-free conditions, H-21-Abeta (Abeta(-/-),T-cell receptor beta (TCRbeta(-/-)), and recombinase-activating gene 1 (RAG-1(-/-) gene disruption mutant mice, deficient in conventional CD4+ T cells, TCRalphabeta cells, and all peripheral T and B lymphocytes, respectively, consistently developed lethal Pneumocystis carinii pneumonia through natural infection. The most severe symptoms appeared in RAG-1(-/-) mutants. In contrast, TCRdelta(-/-) and beta2-microglobulin(-/-)(beta2m-/-) mutants, deficient in TCRgammadelta cells and conventional CD8alphabeta+ TCRalphabeta cells, respectively, were fully resistant to infection. Our data indicate not only the insufficiency but also the dispensability of CD8 alphabeta+TCRalphabeta cells and of TCRgammadelta lymphocytes in resistance to P. carinii infection. Under disease conditions, large numbers of unusual single-positive CD4+ and CD8alphabeta+ as well as double-negative TCRgammadelta subpopulations of cells accumulated in lungs of TCRbeta(-/-) mutants. This accumulation was consistently accompanied by a drastic increase in the pulmonary B-cell population. In contrast, CD8alphabeta+ TCR alpha beta cells, but no B cells, appeared in lungs of parasitized Abeta (-/-) mutants. Since lung damage and parasite numbers were less prominent in morbid TCRbeta(-/-) and Abeta(-/-) mutants than in diseased RAG-1(-/-) mice, the remaining lymphocytes accumulating in lungs of the former two mutants seem to perform residual resistance functions.     

Decreased surfactant protein-B expression and surfactant dysfunction in a murine model of acute lung injury

This study examines the relationships between inflammation, surfactant protein (SP) expression, surfactant function, and lung physiology in a murine model of acute lung injury (ALI). 129/J mice received aerosolized endotoxin lipopolysaccharide [LPS] daily for up to 96 h to simulate the cytokine release and acute inflammation of ALI. Lung elastance (E(L)) and resistance, lavage fluid cell counts, cytokine levels, phospholipid and protein content, and surfactant function were measured. Lavage and lung tissue SP content were determined by Western blot and immunohistochemistry, and tissue messenger RNA (mRNA) levels were assessed by Northern blot and in situ hybridization. Tumor necrosis factor-alpha and neutrophil counts in bronchoalveolar lavage fluid increased within 2 h of LPS exposure, followed by increases in total protein, interleukin (IL)-1beta, IL-6, and interferon-gamma. E(L) increased within 24 h of LPS exposure and remained abnormal up to 96 h. SP-B protein and mRNA levels were decreased at 24, 48, and 96 h. By contrast, SP-A protein and mRNA levels and SP-C mRNA levels were not reduced. Surfactant dysfunction occurred coincident with changes in SP-B levels. This study demonstrates that lung dysfunction in mice with LPS-ALI corresponds closely with abnormal surfactant function and reduced SP-B expression.     

Phosphatidylcholine molecular species in lung surfactant: composition in relation to respiratory rate and lung development.

Surfactant reduces surface tension at the air-liquid interface of lung alveoli. While dipalmitoylphosphatidylcholine (PC16:0/ 16:0) is its main component, proteins and other phospholipids contribute to the dynamic properties and homeostasis of alveolar surfactant. Among these components are significant amounts of palmitoylmyristoylphosphatidylcholine (PC16:0/ 14:0) and palmitoylpalmitoleoylphosphatidylcholine (PC16:0/ 16:1), whereas in surfactant from the rigid tubular bird lung, PC16:0/14:0 is absent and PC16:0/16:1 strongly diminished. We therefore hypothesized that the concentrations of PC16:0/14:0 and PC16:0/16:1 in surfactants correlate with differences in the respiratory physiology of mammalian species. In surfactants from newborn and adult mice, rats, and pigs, molar fractions of PC16:0/14:0 and PC16:0/16:1 correlated with respiratory rate. Labeling experiments with [methyl-(3)H]choline in mice and perfused rat lungs demonstrated identical alveolar proportions of total and newly synthesized PC16:0/14:0, PC16:0/16:1, and PC16:0/16:0, which were much higher than those of other phosphatidylcholine species. In surfactant from human term and preterm neonates, fractional concentrations not only of PC16:0/16:0 but also of PC16:0/14:0 and PC16:0/ 16:1 increased with maturation. Our data emphasize that PC16:0/14:0 and PC16:0/16:1 may be important surfactant components in alveolar lungs, and that their concentrations are adapted to respiratory physiology.     

Intracellular oxidant production and cytokine responses in lung macrophages: evaluation of fluorescent probes

The fluorescent probes dichlorofluorescin (DCFH), dihydrorhodamine (DHR), and hydroethidine (HE) allow convenient assay of alveolar macrophage (AM) oxidant responses to enviromental particulates and pathogens. We sought to more precisely define the relationship of these measures of oxidant stress to production of pro-inflammatory cytokines. Normal AMs were challenged in vitro with a panel of soluble or particulate stimuli in the presence of DCFH, HE, or DHR. Flow cytometry measured cell-associated fluorescence and relative particle uptake. Tumor necrosis factor alpha and macrophage inflammatory protein 2 expression were quantitated in the same experiments. We observed variable and complex correlations between intracellular oxidant production as reported by these probes and subsequent cytokine response, including examples of striking discordance (e.g., lipopolysaccharide induced large cytokine responses with minimal probe oxidation, whereas fly ash particles caused marked oxidation of DCFH but trivial TNF release; TiO2 caused oxidation of DHR and HE, but not DCFH, and also did not increase cytokine production). Although fluorescent probes offer many advantages in analysis of intracellular oxidant responses, the data indicate that they cannot be used reliably as quantitative predictors of AM cytokine responses to environmental particulates or other stimuli.