Pneumocystis carinii β-Glucan Induces Release of Macrophage Inflammatory Protein-2 from Primary Rat Alveolar Epithelial Cells via a Receptor Distinct from CDllb/CD18
Pneumocystis carinii (PC) pneumonia remains a life threatening nfection in immunocompromised hosts. Neutrophilic inflammation and diffuse alveolar damage contribute significantly to respiratory failure characteristic of severe PC pneumonia.
We have recently shown that alveolar macrophages produce tumor necrosis factor (TNF) and macrophage inflammatory protein-2 (MIP-2) in response to purified particulate PC β-glucan [1]. Although several different receptors have been reported to bind β-glucan, the best studied among is the CDllb/CD18 complex (CR3). The membrane glycosphingolipid, lactosylceramide (CDwl7), has also been shown to participate in the glucan receptor complex [2].
Recent studies have demonstrated that alveolar epithelial cells (AEC's) play an important role in lung inflammation by the release of cytokines and chemokines. The purpose of this study is to determine if purified particulate PC β-glucan induces MIP-2 expression from primary AEC's.
MATERIAL AND METHODS
AEC's were isolated from Sprague-Dawley rats according to the method of Dobbs [3]. PC P-glucan was generated from isolated PC organisms from immunosuppressed Sprague-Dawley rats as we have previously described.[l] To characterize the AEC's response to PC β-glucan, varying concentrations (1-10 × 107 particles/ml) of PC β-glucan were used to stimulate AECs for 6 hours. An optimal concentration (5×106) was chosen and AEC's stimulated for 0 through 24 hrs and MIP-2 determined by ELISA.
Ribonuclease protection assay (RPA) was used to determine MIP-2 mRNA levels after glucan challenge. AEC's (5 × 106 cells/well) or MLE-12 cells (5 × 106 cells/well, ATCC) were were stimulated with PC P-glucan (5 × 106 particles/ml) for 0, 2, 6, and 24 hours. RNA was isolated using the RNeasy Mini Kit (Qiagen). Ribonuclease protection assay (RPA) was then performed using T7 polymerase and [P32]-radiolabelled, antisense riboprobes for rat MIP-2, MCP-1, IL-la, TNFa, TNFβ, IL-3, IL-4, IL-5, II-10, IL-2, IFNγ, L32, and GAPDH. RNase protected probes were run on 6% polyacrylamide gels for 20 minutes at 1200 volts. The gels were then dried and exposed to X-ray film.
We next carried out blocking studies to investigate potential receptors to which glucan binds AEC's. Isolated AEC's were incubated for 1hr with the soluble glucans laminarin (1mg/ml), or pustulan (1mg/ml), or antibodies directed against CDllb (M-19, Santa Cruz), CDw17 (Huly-13, Ancell), digested CDwl7, or Asialo-GMl (Matreya) before stimulation with β-glucan. MIP-2 concentrations were measured by ELISA. To determine if CD11b is present on AEC's, we also performed immunoprecipitation/Western blots using anti-CD11b.
To further study CDwl7's role in the glucan receptor complex, anti-CDwl7 was pre-incubated with solubilized lactosylceramide and then added to AEC's to determine if this would reverse the effects of blocking CDwl7. AEC's were also incubated with the glycosphingolipid synthesis inhibitor, N-butyldeoxyjirimycin (N-BDJ), and lipid free media. AEC's were then stimulated with β-glucan to determine if lipid depletion would cause a decrease in AEC's MIP-2 response.
AEC's increased MIP-2 production in response to P. cariniiβ-glucan in a dose and time dependent fashion. The MIP-2 levels after PC β-glucan stimulation were significantly increased as early as 2 hours post stimulation with maximal levels at 16 and 24 hours. MIP-2 mRNA was also significantly increased after PC β-glucan stimulation in AEC's and MLE-12 cells, an alveolar cell line.
CDllb/CD18 (CR3) is the best studied of the β-glucan receptors. Imunoprecipitation and western blot, however, clearly demonstrated that CD1lb is not present on the AEC's. Incubation of cells with anti-CDllb before glucan stimulation furthermore did not demonstrate any decrease in MIP-2 secretion
Blocking studies did demonstrate that incubation of AEC's with anti-CDwl7 before β-glucan stimulation results in a significant decrease in MIP-2 production. Incubation of AEC's with digested anti-CDwl7 fragments (IgG, rIgG) resulted in a similar level of MIP-2 attenuation compared to the whole IgM molecule ruling out steric blocking. Preincubation of anti-CDwl7 and solubilized lactosylceramide reversed this effect. Incubation of AEC's with the glycosphingolipid synthesis inhibitor, N-BDJ [4], prior to glucan stimulation resulted in a significant decrease in MIP-2 release.
How CDwl7 transmits its signal has yet to be elucidated. Glycosphingolipids are concentrated in microdomains on the plasma membrane surface. These microdomains or “rafts” are thought to be platforms where signaling molecules are concentrated [5]. Interestingly, antibodies to asialoGMl, another glycosphingolipid on AECs and a known Pseudomonas pilin receptor, did not attenuate MIP-2 production.
Our study demonstrates that the β-glucan component of the Pneumocystis carinii cell wall is able to stimulate alveolar epithelial cells to produce MIP-2. Our study further demonstrates that the membrane glycosphingolipid, lactosylceramide, likely participates in the receptor complex for PC β-glucan on AEC's. The MIP-2 produced by AECs may contribute significantly to the neutrophilic lung inflammation that characterizes severe Pneumocystis pneumonia.
