Dennis R. Voelker

By Binding SIRPα or Calreticulin/CD91, Lung Collectins Act as Dual Function Surveillance Molecules to Suppress or Enhance Inflammation

Surfactant proteins A and D (SP-A and SP-D) are lung collectins composed of two regions, a globular head domain that binds PAMPs and a collagenous tail domain that initiates phagocytosis. We provide evidence that SP-A and SP-D act in a dual manner, to enhance or suppress inflammatory mediator production depending on binding orientation. SP-A and SP-D bind SIRPalpha through their globular heads to initiate a signaling pathway that blocks proinflammatory mediator production. In contrast, their collagenous tails stimulate proinflammatory mediator production through binding to calreticulin/CD91. Together a model is implied in which SP-A and SP-D help maintain a non/anti-inflammatory lung environment by stimulating SIRPalpha on resident cells through their globular heads. However, interaction of these heads with PAMPs on foreign organisms or damaged cells and presentation of the collagenous tails in an aggregated state to calreticulin/CD91, stimulates phagocytosis and proinflammatory responses.

120-kD surface glycoprotein of Pneumocystis carinii is a ligand for surfactant protein A.

Pneumocystis carinii is the most common cause of life-threatening pneumonia in immunocompromised patients. In the current study, surfactant protein A (SP-A), the major nonserum protein constituent of pulmonary surfactant, is demonstrated to bind P. carinii in a specific and saturable manner. SP-A is surface bound and does not appear to be internalized or degraded by the P. carinii organism. Furthermore, SP-A binding to P. carinii is time- and calcium-dependent and is competitively inhibited by mannosyl albumin. In the absence of calcium or the presence of excess mannosyl albumin, SP-A binding to P. carinii is reduced by 95 and 71%, respectively. SP-A avidly binds P. carinii with a Kd of 8 x 10(-9) M and an estimated 8.4 x 10(6) SP-A binding sites per P. carinii organism, as determined from Scatchard plots. SP-A is shown to bind P. carinii in vivo, and a putative binding site for SP-A on P. carinii is demonstrated to be the mannoserich surface membrane glycoprotein gp120. These findings suggest that P. carinii can interact with the phospholipid-rich material in the alveolar spaces by specifically binding a major protein constituent of pulmonary surfactant.

Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7.

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.

Surfactant Proteins A and D Bind CD14 by Different Mechanisms

Surfactant proteins A (SP-A) and D (SP-D) are lung collectins that are constituents of the innate immune system of the lung. Recent evidence (Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D. R., and Kuroki, Y. (1999) J. Immunol. 163, 387–395) demonstrates that SP-A modulates lipopolysaccharide (LPS)-induced cellular responses by direct interaction with CD14. In this report we examined the structural elements of the lung collectins involved in CD14 recognition and the consequences for CD14/LPS interaction. Rat SP-A and SP-D bound CD14 in a concentration-dependent manner. Mannose and EDTA inhibited SP-D binding to CD14 but did not decrease SP-A binding. The SP-A binding to CD14 was completely blocked by a monoclonal antibody that binds to the SP-A neck domain but only partially blocked by an antibody that binds to the SP-A lectin domain. SP-A but not SP-D bound to deglycosylated CD14. SP-D decreased CD14 binding to both smooth and rough LPS, whereas SP-A enhanced CD14 binding to rough LPS and inhibited binding to smooth LPS. SP-A also altered the migration profile of LPS on a sucrose density gradient in the presence of CD14. From these results, we conclude that 1) lung collectins bind CD14, 2) the SP-A neck domain and SP-D lectin domain participate in CD14 binding, 3) SP-A recognizes a peptide component and SP-D recognizes a carbohydrate moiety of CD14, and 4) lung collectins alter LPS/CD14 interactions.

Surfactant protein A and surfactant protein D in health and disease

Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung. Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin. Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules. SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin. Initially, most research interest focused on its role in surfactant homeostasis. Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells. SP-D is much less involved with the surfactant system. SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin. Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis. In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease. The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo.Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung. Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin. Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules. SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin. Initially, most research interest focused on its role in surfactant homeostasis. Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells. SP-D is much less involved with the surfactant system. SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin. Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis. In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease. The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo.

Pulmonary Surfactant Protein A Up-Regulates Activity of the Mannose Receptor, a Pattern Recognition Receptor Expressed on Human Macrophages

Inhaled particulates and microbes are continually cleared by a complex array of lung innate immune determinants, including alveolar macrophages (AMs). AMs are unique cells with an enhanced capacity for phagocytosis that is due, in part, to increased activity of the macrophage mannose receptor (MR), a pattern recognition receptor for various microorganisms. The local factors that “shape” AM function are not well understood. Surfactant protein A (SP-A), a major component of lung surfactant, participates in the innate immune response and can enhance phagocytosis. Here we show that SP-A selectively enhances MR expression on human monocyte-derived macrophages, a process involving both the attached sugars and collagen-like domain of SP-A. The newly expressed MR is functional. Monocyte-derived macrophages on an SP-A substrate demonstrated enhanced pinocytosis of mannose BSA and phagocytosis of Mycobacterium tuberculosis lipoarabinomannan-coated microspheres. The newly expressed MR likely came from intracellular pools because: 1) up-regulation of the MR by SP-A occurred by 1 h, 2) new protein synthesis was not necessary for MR up-regulation, and 3) pinocytosis of mannose BSA via MR recycling was increased. AMs from SP-A−/− mice have reduced MR expression relative to SP-A+/+. SP-A up-regulation of MR activity provides a mechanism for enhanced phagocytosis of microbes by AMs, thereby enhancing lung host defense against extracellular pathogens or, paradoxically, enhancing the potential for intracellular pathogens to enter their intracellular niche. SP-A contributes to the alternative activation state of the AM in the lung.

Identification and Characterization of the Major Lysophosphatidylethanolamine Acyltransferase in Saccharomyces cerevisiae

We recently demonstrated that yeast actively import lysophosphatidylethanolamine (lyso-PtdEtn) through the action of plasma membrane P-type ATPases and rapidly acylate it to form PtdEtn. The predominant lyso-PtdEtn acyltransferase (LPEAT) activity present in cellular extracts is acyl-CoA dependent, but the identity of the gene encoding this activity was unknown. We now demonstrate that a previously uncharacterized open reading frame, YOR175C, encodes the major acyl-CoA-dependent LPEAT activity in yeast and henceforth refer to it as ALE1 (acyltransferase for lyso-PtdEtn). Ale1p is an integral membrane protein and is highly enriched in the mitochondria-associated endoplasmic reticulum membrane. It is a member of the membrane-bound O-acyltransferase family and possesses a dibasic motif at its C terminus that is likely responsible for Golgi retrieval and retention in the endoplasmic reticulum. An ale1Δ strain retains only trace amounts of acyl-CoA-dependent LPEAT activity, and strains lacking the capacity for PtdEtn synthesis via the phosphatidylserine decarboxylase and Kennedy pathways show a stringent requirement for both exogenous lyso-PtdEtn and a functional ALE1 gene for viability. Ale1p catalytic activity has a pH optimum between pH 7 and 7.5 and a strong preference for unsaturated acyl-CoA substrates.

Mechanism of binding of surfactant protein D to influenza A viruses: importance of binding to haemagglutinin to antiviral activity.

Collectins are important in the initial containment of a variety of pathogens, including influenza A virus (IAV). We provide the first systematic evaluation of the oligosaccharide-binding sites for pulmonary surfactant protein D (SP-D) on specific IAV coat glycoproteins and define the relationship between this binding and antiviral activity. With the use of several techniques, SP-D was found to bind via its carbohydrate-recognition domain (CRD) to mannosylated, N-linked carbohydrates on the HA(1) domain of the haemagglutinin (HA) and on the neuraminidase of IAV. Using a set of IAV strains that differed in the level and site of glycosylation, and a panel of recombinant collectins, we found that binding of SP-D to the globular domain of the HA was critical in mediating the inhibition of viral haemagglutination activity and infectivity. We also demonstrated that the pattern of binding of a collectin to IAV glycoproteins can be modified by altering the monosaccharide-binding affinity of its CRD or by linking the CRD to a different N-terminal/collagen domain. These studies clarify the mechanisms of viral neutralization by collectins and might be useful in engineering collectins for enhanced antiviral activity.

Regulatory mechanisms of surfactant secretion.

Surfactant secretion is a critical regulated process in the metabolism of pulmonary surfactant. Presumably, because this process is vital to the survival of the organism, there are several independent pathways for stimulating secretion which work through different cell surface receptors and signaling mechanisms. In addition, there is apparent homeostatic regulation in that two components of surfactant, namely SP-A and dipalmitoylphosphatidylcholine, can inhibit secretion. Although secretion of surfactant has been studied for over two decades, there remains some important issues to be resolved. In vivo secretion can be stimulated by hyperventilation or even a single large breath. However, we do not know the biochemical mechanism for this physiologically important form of stimulation. In vitro, we know many of the proximal events in signaling, but we do not know how the lamellar bodies move within a cell or the docking mechanism at the plasma membrane. Many investigators have demonstrated that SP-A will inhibit secretion in vitro, but the mechanism is not known. Finally, there is a route of secretion of SP-A independent of lamellar bodies, but we do not know details of this pathway nor its regulation.

Phosphatidylethanolamine Has an Essential Role inSaccharomyces cerevisiae That Is Independent of Its Ability to Form Hexagonal Phase Structures

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides ∼90% of total cellular phosphatidylserine decarboxylase activity. When thePSD1 gene is deleted, the resultant strain(psd1Δ) grows normally at 30 °C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37 °C) or on the nonfermentable carbon source lactate, the growth of psd1Δ strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Δ psd2Δ dpl1Δ strain can only grow in the presence of ethanolamine. In contrast, a psd1Δ psd2Δstrain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Δ psd2Δ cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by31P NMR.