Primary alveolar epithelial cell surface membrane microdomain function is required for Pneumocystis β-glucan-induced inflammatory responses

Abstract

Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent β-(1→3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-α and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.

Figure 1. Disruption of microdomain function impairs P. carinii β-glucan-induced inflammatory cytokine expression

In order to determine the effect of various internalization mechanisms, primary AECs were challenged with PCBG with or without inhibitory pretreatment. To impair general internalization, cells were maintained at 4°C for 30 minutes prior to challenge. Microdomain function was disrupted by several mechanisms, including 30 minutes pretreatment with genistein, methyl-β-cyclodextran or nystatin. In contrast, internalization via clathrin-mediated mechanisms was inhibited by 30 minutes pretreatment with chlorpromazine. ELISA detection of MIP-2 was performed on the supernatant after four hours of challenge. These experiments showed significant suppression of PCBG-induced MIP-2 expression following both cold and anti-microdomain pretreatments, while anti-clathrin pretreatment had little effect. (* Denotes P<0.05).

Figure 2. P. carinii β-glucan induces microdomain-localized protein kinase C activation

Primary AECs were harvested from monolayer with or without PCBG challenge. Lipid microdomains (rafts) were then isolated from the samples by Triton X-100 fractionation. Following homogenization, the samples were separated by SDS-PAGE then submitted to Western blotting using a pan-phospho-PKC antibody. Samples were exposed to HPR-conjugated secondary antibody and ECL chemiluminescent detection system, prior to autoradiography. While the unchallenged cell samples showed little activated (phosporylated) PKC, the PCBG-challenged cells demonstrated a significant increase in the autoradiographic signal. Double bands are noted, due to multiple PKC isoforms. This effect appeared maximal at 15 minutes and began to decline around 30 minutes.

Figure 3. Alveolar epithelial cells internalize fluorescently labeled P. carinii β-glucan

After approximately 48 hours culture, primary AECs were exposed to DTAF-labeled PCBG for one hour. The samples were then acid stripped, formalin fixed, and treated with a lipophilic counterstain prior to being submitted to confocal fluorescent microscopy. Multiple images in the z-plane were obtained to confirm complete internalization of PCBG particle. This photomicrograph demonstrates DTAF-labeled PCBG (green) in various stages of internalization. The internalized PCBG displayed yellow coloration when imaged in this manner.

Figure 4. Microdomain disruption impairs internalization of DTAF-labeled P. carinii β-glucan by alveolar epithelial cells

Primary AECs were challenged with DTAF-labeled PCBG with or without pretreatment, and then submitted to conventional fluorescence microscopy. A. DTAF-labeled PCBG was internalized by alveolar epithelial cells 2 hours after challenge at 37°C. B. In contrast, culture of the AECs at 4°C prevented PCBG uptake. C. Monoclonal antibodies targeting the microdomain glycosphingolipid constituent lactosylceramide also prevented PCBG uptake. D. However, preincubation of the anti-lactosylceramide antibody with free lactosylceramide abrogated this effect. E. Finally, disruption of lipid microdomains with nystatin also inhibited PCBG uptake.

Figure 5. Impaired microdomain function decreases internalized DTAF-labeled P. carinii β-glucan signal

Primary rat AECs were challenged with DTAF-labeled PCBG with or without pretreatment by a variety of inhibitors of internalization. After four hours of challenge, the samples were acid stripped, then lysed with sodium hydroxide. Homogenized equal volumes from each sample were then assayed by conventional photospectrofluorimetry. Anti-lactosylceramide antibodies and nystatin both significantly impaired the internalization of DTAF-labeled PCBG, as measured by this method. Cytochalasin D had a lesser, but still significant, effect. Antibodies against an arbitrary glycosphingolipid (GM-1) had no effect on PCBG uptake. (* Denotes P<0.01).