Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in a variety of clinical settings. An early step in Pneumocystis infection involves the attachment of organisms to alveolar epithelial cells (AECs). AECs produce chemokines in response to Pneumocystis stimulation, but the upstream host-pathogen interactions that activate AEC signaling cascades are not well-defined. MyD88 is an adaptor molecule required for activation of proinflammatory signaling cascades following Toll-like receptor (TLR)-dependent recognition of conserved molecular patterns on pathogens. To determine whether the TLR/MyD88 pathway is required for the AEC chemokine response to Pneumocystis, wild-type (WT) and MyD88-deficient AECs were incubated with Pneumocystis. As expected, WT AECs produced CCL2 and CXCL2 following Pneumocystis stimulation. In contrast, MyD88-deficient AECs were severely impaired in their ability to respond to Pneumocystis. MyD88-deficient AECs did not display Pneumocystis-induced Jun N-terminal protein kinase activation and produced much less chemokine than Pneumocystis-stimulated WT AECs. Using a panel of TLR agonists, primary murine AECs were found to respond vigorously to TLR2 and TLR4 agonists. However, the AEC chemokine response to Pneumocystis did not require TLR2 or TLR4. Surprisingly, the interleukin-1 receptor (IL-1R) was required for an AEC chemokine response to Pneumocystis. The role of MyD88 in early responses during Pneumocystis infection was supported by in vivo studies demonstrating that MyD88-deficient mice showed impaired Pneumocystis-stimulated chemokine production and impaired inflammatory cell recruitment. These data indicate an important role for MyD88 in the AEC inflammatory response to Pneumocystis.
Pneumocystis is an opportunistic fungal respiratory pathogen that causes life-threatening pneumonia (Pcp) in patients suffering from defects in cell-mediated immunity, including those with acquired immunodeficiency syndrome (AIDS) and immunosuppression secondary to chemotherapy or organ transplantation. Despite major advances in health care, the mortality associated with Pcp has changed little over the past 25 years. Pcp remains a leading cause of death among HIV infected patients, with mortality rates of 50% or higher for patients developing severe Pcp. In addition, as more potent immunosuppressive therapies are developed for chronic inflammatory diseases, more cases of Pcp are occurring in non-HIV patients and in previously unreported clinical settings. These features highlight the importance of developing a better understanding of the pathogenesis of this disease, and the need to search for new therapeutic strategies to improve the outcome of Pcp patients. Immune-mediated inflammatory responses play an important role in the pathogenesis of Pcp, and may be even more significant in determining the outcome of Pcp than direct damage due to the organism itself. In this review we will summarize the immunopathogenic mechanisms that contribute to Pcp-associated lung injury, and discuss the potential to target these pathways for adjunctive immune modulation therapy for Pcp.
The T cell-mediated immune response elicited by Pneumocystis plays a key role in pulmonary damage and dysfunction during Pneumocystis pneumonia (PcP). Mice depleted of CD4+ and CD8+ T cells prior to infection are markedly protected from PcP-related respiratory deficit and death despite progressive lung infection. However, the therapeutic effectiveness of antibody-mediated disruption of T cell function in mice already displaying clinical symptoms of disease has not been determined. Therefore, a murine model of PcP-related immune reconstitution inflammatory syndrome was used to assess whether antibody to the pan-T cell molecule CD3 is effective for reducing the severity of PcP when administered after the onset of disease. Mice that received anti-CD3 antibody exhibited a rapid and dramatic halt in the PcP-associated pulmonary function decline within one week post-treatment, and a striking enhancement of survival rate compared to mice receiving control antibody. Physiological improvement in anti-CD3 treated mice was associated with a significant reduction in the number of CD4+ and CD8+ T cells recovered in lung lavage fluid. This effectiveness of anti-CD3 was noted whether or not the mice also received antibiotic therapy with trimethoprim-sulfamethoxazole. These data suggest that monoclonal antibody-mediated disruption of T cell function may represent a specific and effective adjunctive therapy to rapidly reverse the ongoing pathological immune response occurring during active PcP. Thus, the anti-human CD3 monoclonal antibody OKT3, which is already in clinical use, has the potential to be developed as an adjunctive therapy for PcP.
P. carinii pneumonia; Anti-CD3; Inflammation; Lymphocytes
Although T cells are critical for host defense against respiratory fungal infections, they also contribute to the immunopathogenesis of Pneumocystis pneumonia (PcP). However, the precise downstream effector mechanisms by which T cells mediate these diverse processes are undefined. In the current study the effects of immune modulation with sulfasalazine were evaluated in a mouse model of PcP-related Immune Reconstitution Inflammatory Syndrome (PcP-IRIS). Recovery of T cell-mediated immunity in Pneumocystis-infected immunodeficient mice restored host defense, but also initiated the marked pulmonary inflammation and severe pulmonary function deficits characteristic of IRIS. Sulfasalazine produced a profound attenuation of IRIS, with the unexpected consequence of accelerated fungal clearance. To determine whether macrophage phagocytosis is an effector mechanism of T cell-mediated Pneumocystis clearance and whether sulfasalazine enhances clearance by altering alveolar macrophage phagocytic activity, a novel multispectral imaging flow cytometer-based method was developed to quantify the phagocytosis of Pneumocystis in vivo. Following immune reconstitution, alveolar macrophages from PcP-IRIS mice exhibited a dramatic increase in their ability to actively phagocytose Pneumocystis. Increased phagocytosis correlated temporally with fungal clearance, and required the presence of CD4+ T cells. Sulfasalazine accelerated the onset of the CD4+ T cell-dependent alveolar macrophage phagocytic response in PcP-IRIS mice, resulting in enhanced fungal clearance. Furthermore, sulfasalazine promoted a TH2-polarized cytokine environment in the lung, and sulfasalazine-enhanced phagocytosis of Pneumocystis was associated with an alternatively activated alveolar macrophage phenotype. These results provide evidence that macrophage phagocytosis is an important in vivo effector mechanism for T cell-mediated Pneumocystis clearance, and that macrophage phenotype can be altered to enhance phagocytosis without exacerbating inflammation. Immune modulation can diminish pulmonary inflammation while preserving host defense, and has therapeutic potential for the treatment of PcP-related immunopathogenesis.
Pneumocystis is a fungal respiratory pathogen that causes life-threatening pneumonia (PcP) in immunosuppressed patients. PcP remains an infectious complication of AIDS and cancer, and is emerging in previously unrecognized clinical settings. Despite dramatic advances in health care and the availability of antibiotics to treat this infection, mortality rates have improved little over the past 25 years. T cell-mediated immunity is critical for host defense against respiratory fungal infections. However, T cells also cause PcP-related inflammation and lung injury. The results of the current study indicate that the immune response to Pneumocystis can be modulated to reduce tissue damaging inflammation while enhancing anti-fungal host defense. Alveolar macrophages recognize and eliminate pathogens from the lung and also regulate inflammation. We have identified alveolar macrophages as the effector cells for T cell-dependent clearance of Pneumocystis from the lung, and demonstrated that macrophage phenotype can be altered to enhance microbe elimination without promoting inflammatory injury. These results suggest that the effector mechanism of T cell-mediated fungal clearance is distinct from the effector mechanism of T cell-mediated lung inflammation and injury. This conceptual advance can be exploited to develop more effective therapeutic strategies to block inflammation while preserving host defense.
The opportunistic organism Pneumocystis carinii (Pc) produces a life-threatening pneumonia (PcP) in patients with low CD4+ T cell counts. Animal models of HIV-AIDS-related PcP indicate that development of severe disease is dependent on the presence of CD8+ T cells and the tumor necrosis factor receptors (TNFR), TNFRsf1a and TNFRsf1b. To distinguish roles of parenchymal and hematopoietic cell TNF signaling in PcP-related lung injury, murine bone marrow transplant chimeras of wild-type, C57Bl6/J, and TNFRsf1a/1b double null origin were generated, CD4+ T-cell depleted, and inoculated with Pc. As expected, C57→C57 chimeras (donor marrow→recipient) developed significant disease as assessed by weight loss, impaired pulmonary function (lung resistance and dynamic lung compliance) and inflammatory cell infiltration. In contrast, TNFRsf1a/1b-/-→TNFRsf1a/1b-/- mice were relatively mildly affected despite carrying the greatest organism burden. Mice solely lacking parenchymal TNFRs (C57→TNFRsf1a/1b-/-) had milder disease than C57→C57 mice. Both groups of mice with TNFR deficient parenchymal cells had low BALF total cell counts and fewer lavagable CD8+ T cells than C57→C57 mice, suggesting that parenchymal TNFR signaling contributes to PcP-related immunopathology through the recruitment of damaging immune cells. Interestingly, mice with WT parenchymal cells but TNFRsf1a/1b-/- hematopoietic cells (TNFRsf1a/1b-/-→C57) displayed exacerbated disease characterized by increased MCP-1 and KC production in the lung and increased macrophage and lymphocyte numbers in the lavage, indicating a dysregulated immune response. This study supports a key role of parenchymal cell TNFRs in lung injury induced by Pc and a potential protective effect of receptors on radio-sensitive, marrow derived cells.
cytokine receptors; fungal; immunodeficiency disease; lung
While CD8+ cells have been shown to contribute to lung injury during Pneumocystis carinii pneumonia (PCP), there are conflicting reports concerning the ability of CD8+ cells to kill P. carinii. To address these two issues, we studied the effect of the presence of CD8+ cells in two mouse models of PCP. In the reconstituted SCID mouse model, depletion of CD8+ cells in addition to CD4+ cells after reconstitution did not result in increased numbers of P. carinii cysts compared to the numbers of cysts in mice with only CD4+ cells depleted. This result was observed regardless of whether the mice were reconstituted with naïve or P. carinii-sensitized lymphocytes. In contrast, reconstitution with sensitized lymphocytes resulted in more rapid onset of lung injury that was dependent on the presence of CD8+ cells. The course of organism replication over a 6-week period was also examined in the CD4+-T-cell-depleted and CD4+- and CD8+-T-cell-depleted mouse model of PCP. Again, the organism burdens were identical at all times regardless of whether CD8+ cells were present. Thus, in the absence of CD4+ T cells, CD8+ T cells are a key contributor to the inflammatory lung injury associated with PCP. However, we were unable to demonstrate an in vivo effect of these cells on the course of P. carinii infection.
Pneumocystis carinii is an opportunistic fungal pathogen that causes P. carinii pneumonia (PCP) in the immunocompromised host. We investigated the role of antibody Fc-mediated function in passive prophylaxis against the development of PCP in SCID mice. By comparison of anti-mouse P. carinii immunoglobulin G1 monoclonal antibody (MAb) 4F11(G1) and its F(ab′)2 derivative in an intranasal immunoprophylaxis model, we determined that Fc-mediated function is required for maximum effect of this antibody. Comparison of efficacy of antibody prophylaxis in SCID mice depleted of complement to that in nondepleted mice demonstrated that complement fixation by MAb 4F11(G1) is also necessary for optimal effect of passively administered antibody, although residual protection was observed in complement-depleted SCID mice. The necessity of complement for optimal PCP prophylaxis by MAb 4F11(G1) suggests that complement may play a role in antibody-mediated protection against development of PCP.
Mice immunized with recombinant mouse Pneumocystis carinii antigen A12-thiredoxin fusion protein developed an antibody response that recognized P. carinii antigens, as determined by Western blotting and immunofluorescence analysis. Compared to mice immunized with thioredoxin alone, mice immunized with A12-thioredoxin had significantly reduced lung P. carinii burdens after CD4+ T-cell depletion and challenge with P. carinii.
Pneumocystis carinii pneumonia (PcP) is a clinically important infection of immunocompromised patients. Although the interaction of Pneumocystis with the alveolar epithelium has been well documented, very little information regarding the epithelial response to Pneumocystis is currently available. In order to study Pneumocystis-epithelium interactions, a murine cell line derived specifically from an alveolar epithelial cell (AEC) was utilized. The coculture of murine AECs with mouse Pneumocystis induced a dose- and time-dependent release of the CXC chemokine MIP-2. Importantly, the specific removal of Pneumocystis from the preparation, or the pretreatment of AECs with sulfasalazine, a potent and specific inhibitor of NF-κB, nearly completely abrogated the chemokine response to Pneumocystis. Since the murine MIP-2 promoter contains consensus κB binding sequences, the ability of Pneumocystis to stimulate NF-κB signaling in AECs was examined. Pneumocystis stimulation of an AEC line stably transfected with a κB-dependent reporter construct triggered the NF-κB signaling pathway and reporter production. These data were confirmed in gel shift assays, providing direct evidence that Pneumocystis induced the nuclear translocation of the p50/p65 heterodimeric form of NF-κB. Maximal NF-κB activation was dependent upon direct contact with viable Pneumocystis organisms. These data demonstrate that Pneumocystis activates NF-κB signaling in AECs and establish a reporter cell line for studying NF-κB activation in AECs. Given the global regulatory functions of the NF-κB family, these findings suggest that Pneumocystis directly alters AEC gene expression in a manner that promotes pulmonary immune and inflammatory responses.
Hoechst 33258 is a compound that binds nucleic acids. We report that Hoechst 33258 exhibits antimicrobial activity against Pneumocystis carinii f. sp. muris in a mouse model for P. carinii pneumonia and against Candida albicans and Candida dubliniensis in vitro. Relative to saline treatment, a 14-day, daily treatment of mice with 37.5 mg of Hoechst 33258/kg of body weight after inoculation with P. carinii reduced by about 100-fold the number of P. carinii organisms detected by either PCR or by microscopy after silver staining. For comparison, treatment based on a dose of 15 to 20 mg of the trimethoprim component in trimethoprim-sulfamethoxazole/kg reduced the number of P. carinii by about fourfold. In vitro inhibition of P. carinii group I intron splicing was observed with a 50% inhibitory concentration (IC50)of 30 μM in 2 or 4 mM Mg2+, suggesting RNA as a possible target. However, Hoechst 33258 inhibits growth of Candida strains with and without group I introns. IC50s ranged from 1 to 9 μM for strains with group I introns and were 12 and 32 μM for two strains without group I introns. These studies demonstrate that compounds that bind fungal nucleic acids have the potential to be developed as new therapeutics for Pneumocystis and possibly other fungi, especially if they could be directed to structures that are not present in mammalian cells, such as self-splicing introns.
Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to infection with Pneumocystis. We have previously shown that there is a significant delay in clearance of the organisms from the lungs of neonatal mice compared to adults. Since alveolar macrophages are the effector cells responsible for killing and clearance of Pneumocystis, we have examined alveolar macrophage activity in neonatal mice. We found that alveolar macrophage activation is delayed about 1 week in Pneumocystis-infected neonates compared to adults. Opsonization of the organism by Pneumocystis-specific antibody resulted in increased clearance of the organism in neonatal mice; however, there was decreased expression of activation markers on neonatal alveolar macrophages and reduced levels of cytokines associated with macrophage activation. Mice born to immunized dams had significant amounts of Pneumocystis-specific immunoglobulin G in their lungs and serum at day 7 postinfection, whereas mice born to naïve dams had merely detectable levels. This difference correlated with enhanced Pneumocystis clearance in mice born to immunized dams. The increase in specific antibody, however, did not result in significant inflammation in the lungs, as no differences in numbers of activated CD4+ cells were observed. Furthermore, there was no difference in cytokine or chemokine concentrations in the lungs of pups born to immune compared to naïve dams. These findings indicate that specific antibody plays an important role in Pneumocystis clearance from lungs of infected neonates; moreover, this process occurs without inducing inflammation in the lungs.
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.
Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived P. carinii was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of P. carinii derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse P. carinii antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse P. carinii recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen, Streptococcus pneumoniae PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the S. pneumoniae surface, and Western blotting identified a protein in S. pneumoniae lysates consistent with the Mr of PspA. A fragment of the S. pneumoniae PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in P. carinii. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize P. carinii derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against P. carinii.
There has been emerging evidence that immunocompetent hosts can harbor Pneumocystis in their lungs. The purpose of this study was to determine the kinetics of Pneumocystis carinii f. sp. muris infection in adult immunocompetent mice and the host immune response to the organisms. To accomplish this, we exposed adult immunocompetent mice to SCID mice infected with P. carinii f. sp. muris by cohousing. We found that P. carinii f. sp. muris was detectable in the lungs of cohoused immunocompetent mice by PCR by 3 weeks after the beginning of cohousing. At about 4 weeks of cohousing, P. carinii f. sp. muris was readily detectable in the lungs of mice by microscopic techniques. Also at this time, P. carinii f. sp. muris-specific immunoglobulin G was found in the sera of the mice, and CD62low CD4- and CD8-positve T cells accumulated in the lungs. Shortly after this immune response, the P. carinii f. sp. muris organisms were cleared from the lungs. Adult mice cohoused for only 1 week also contained P. carinii f. sp. muris cysts detectable by silver staining at 5 and 6 weeks after the beginning of cohousing. We also found that the P. carinii f. sp. muris organisms grew to greater numbers in the lungs of BALB/c mice than in those of C57BL6 mice. This indicates that immunocompetent hosts develop a mild infection with P. carinii f. sp. muris which resolves in 5 to 6 weeks when there is a detectable immune response to the organism. Once an acquired immune response was initiated, the P. carinii f. sp. muris organisms were quickly eliminated without clinical signs of disease.
Passive antibody immunoprophylaxis is one method used to protect patients against infection if they are unable to mount an adequate active immune response. Topical application of antibody may be effective against infections at mucosal sites. Using a SCID mouse model of Pneumocystis carinii pneumonia, we were able to demonstrate protection against an airborne challenge with P. carinii by intranasal administration of antibody. Immunoglobulin M (IgM) monoclonal antibodies to an epitope shared by mouse and human P. carinii organisms reduced organism numbers by more than 99% under the conditions described. An IgG1 switch variant of one of the IgM monoclonal antibodies was also protective. These experiments provide a model for exploring the utility of this approach in protecting at-risk patients from infection with P. carinii.
During Pneumocystis carinii pneumonia (PCP) in mice, the degree of pulmonary inflammation correlates directly with the severity of lung function deficits. Therefore, studies were undertaken to determine whether the host inflammatory response contributes to PCP-related respiratory impairment, at least in part, by disrupting the pulmonary surfactant system. Protein and phospholipid content and surfactant activity were measured in the lavage fluid of infected mice in either the absence or presence of an inflammatory response. At 9 weeks postinfection with P. carinii, nonreconstituted SCID mice exhibited no signs of pulmonary inflammation, respiratory impairment, or surfactant dysfunction. Lavage fluid obtained from these mice had protein/phospholipid (Pr/PL) ratios (64% ± 4.7%) and minimum surface tension values (4.0 ± 0.9 mN/m) similar to those of P. carinii-free control mice. However, when infected SCID mice were immunologically reconstituted, an intense inflammatory response ensued. Pr/PL ratios (218% ± 42%) and minimum surface tension values (27.2 ± 2.7 mN/m) of the lavage fluid were significantly elevated compared to those of the lavage fluid from infected, nonreconstituted mice (P < 0.05). To examine the specific role of CD8+ T-cell-mediated inflammation in surfactant dysfunction during PCP, mice with defined T-cell populations were studied. P. carinii-infected, CD4+-depleted mice had elevated lavage fluid Pr/PL ratios (126% ± 20%) and elevated minimum surface tension values (16.3 ± 1.0 mN/m) compared to normal mice (P < 0.05). However, when infected mice were additionally depleted of CD8+ cells, Pr/PL ratios were normal and surfactant activity was improved. These findings demonstrate that the surfactant pathology associated with PCP is related to the inflammatory process rather than being a direct effect of P. carinii. Moreover, CD8+ lymphocytes are involved in the mechanism leading to surfactant dysfunction.
The transmission of Pneumocystis carinii from person to person was studied by detecting P. carinii-specific DNA in prospectively obtained noninvasive deep-nasal-swab samples from a child with a documented P. carinii pneumonia (PCP), his mother, two contact health care workers, and 30 hospital staff members who did not enter the patient's room (controls). Nested-DNA amplification was done by using oligonucleotide primers designed for the gene encoding the mitochondrial large subunit rRNA of rat P. carinii (P. carinii f. sp. carinii) that amplifies all forms of P. carinii and internal primers specific for human P. carinii (f. sp. hominis). P. carinii f. sp. hominis DNA was detected in samples from the patient and all of his contacts versus none of the 30 hospital staff members. The results, as previously shown in murine models of P. carinii pneumonia, document that person-to-person transmission of P. carinii is possible. This observation suggests that immunocompromised patients not on PCP prophylaxis should not enter the room of a patient with PCP, and it also raises the question as to whether healthy contacts can transmit the disease to immunocompromised patients at risk.
The clinical severity of Pneumocystis carinii pneumonia (PCP) correlates closely with the appearance of pulmonary markers of inflammation. Therefore, a model system was developed whereby physiological studies could be performed on live mice to determine the extent to which pulmonary inflammation contributes to respiratory impairment during PCP. P. carinii–infected severe combined immunodeficient mice displayed little evidence of pulmonary inflammation and exhibited normal oxygenation and dynamic lung compliance. When comparably infected littermates were immunologically reconstituted, however, an intense immune-mediated inflammatory response was observed that resulted in significant decreases in both lung compliance and oxygenation. As the pneumonia resolved pulmonary function returned toward normal. To begin to define the cell populations contributing to inflammation-associated respiratory impairment during PCP, similar studies were performed in CD4+ T cell–depleted mice. Mice depleted of both CD4+ and CD8+ cells developed infection, but they demonstrated neither abnormal lung compliance nor increased respiratory rate and displayed no markers of lung injury. In contrast, mice depleted of only CD4+ T cells exhibited severe pulmonary inflammation and injury, decreased oxygenation and lung compliance, and increased respirations. Respiratory compromise was associated with the presence of activated CD8+ cells and neutrophils in broncho-alveolar lavage fluid. These observations provide direct experimental evidence that the host’s response to P. carinii directly impairs pulmonary function and contributes to the pathogenesis of PCP. Furthermore, CD8+ T cells likely contribute to the respiratory compromise observed during PCP.
Pneumocystis carinii is an important pulmonary pathogen responsible for morbidity and mortality in patients with AIDS. The acute-phase response (APR), the primary mechanism used by the body to restore homeostasis following infection, is characterized by increased levels of circulating fibrinogen (FBG). Although the liver is the primary site of increased FBG synthesis during the APR, we unexpectedly discovered that FBG is synthesized and secreted by lung alveolar epithelial cells in vitro during an inflammatory stimulus. Therefore, we sought to determine whether lung epithelial cells produce FBG in vivo using animal models of P. carinii pneumonia (PCP). Inflammation was noted by an influx of macrophages to P. carinii-infected alveoli. Northern hybridization revealed that γ-FBG mRNA increased two- to fivefold in P. carinii-infected lung tissue, while RNA in situ hybridization demonstrated increased levels of γ-FBG mRNA in the lung epithelium. Immunoelectron microscopy detected lung epithelial cell-specific production of FBG, suggesting induction of a localized inflammatory response resembling the APR. A systemic APR was confirmed by a two- to fivefold upregulation of the levels of hepatic γ-FBG mRNA in animals with PCP, resulting in a corresponding increase in levels of FBG in plasma. Furthermore, immunoelectron microscopy revealed the presence of FBG at the junction of cell membranes of trophic forms of P. carinii organisms aggregated along the alveolar epithelium. These results implicate FBG in the pathogenesis of PCP in a manner similar to that of the adhesive glycoproteins fibronectin and vitronectin, which are known to participate in intra-alveolar aggregation of organisms and adherence of P. carinii to the lung epithelium.
Immunization with whole Pneumocystis carinii has been shown to protect mice from the development of P. carinii pneumonia (PCP) when they are subsequently immunosuppressed and challenged with viable organisms. To determine whether these results could be duplicated by using a subunit vaccine, we examined the immunogenicity and efficacy of an immunization strategy based on P. carinii gpA. This antigen was chosen for study because passive immunoprophylaxis, based on gpA, has been shown to be partially protective in various animal models of infection. Immunization with gpA produced an anti-gpA specific antibody response comparable to that resulting from immunization with whole organisms. However, in contrast to immunization with whole P. carinii, which was protective, immunization with gpA did not protect T-cell-depleted mice from the development of PCP. These studies suggest that other antigens in addition to gpA need to be evaluated for their role in protective immunity against P. carinii.