Role of macrophage receptor MARCO in host defense

Sammanfattning: Innate immunity, the first line of defense against infectious microorganisms, appeared early in evolution. Innate immunity relies on specialized cells such as macrophages that are the first to encounter pathogens during infection. Macrophages are monocyte-derived cells which express several host-defense receptors which can be divided into two classes; those dependent on opsonizing components for recognizing pathogens, and those that can recognize pathogens directly. This latter class of receptors, pattern-recognition receptors, bind a variety of ligands including mannans and zymosan in the yeast cell wall, and various bacterial cell-wall components, such as lipopolysaccharide (LPS), lipopeptides, lipoteichoic acid (LTA) and peptidoglycans. Class A scavenger receptors are a family of pattern-recognition receptors composed of three members: Scavenger Receptor A (SR-A), Macrophage Receptor with a Collagenous structure (MARCO), and a recently identified protein Scavenger Receptor with C-type Lectin (SRCL). MARCO is a novel member of the class A scavenger receptors. It has a small intracellular N-terminal domain (I), a transmembrane domain (II), an extracellular region with a short spacer (III), a collagenous domain (IV), and a C-terminal cysteine-rich domain (V). In normal mice, this receptor is expressed only by a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes, as well as in the macrophages of the peritoneal cavity. Cells transfected with MARCO cDNA avidly bind both Gram-negative and Gram-positive bacteria, but not yeast. MARCO has also been found to be a major receptor on alveolar macrophages for binding of unopsonized environmental particles, such as TiO2 and Fe2O3. These findings strongly suggest a role for MARCO in host-defense mechanisms. As a first part of this work, we have determined the primary structure of human MARCO and studied its spatial expression using in situ hybridization. Also, the disulfide bridge pattern of the COOH-terminal cysteine-rich domain V was determined. The work in this, and the second part of the study convincingly demonstrated that the cysteine-rich domain V is the predominant bacteria-binding domain in MARCO. Interestingly, the corresponding domain of SR-A is still without a known function. Studies with a series of MARCO truncations containing only short segments of domain V provided information about the requirements for high-affinity bacterial binding. These studies showed that the motif RXR was identified as an essential element for high-affinity bacterial binding in these forms of MARCO. As an important part of this study, we established the production and purification system for recombinant soluble MARCO. Analysis of the structure of the purified protein indicated that soluble MARCO forms stable elongated triple-helical molecules. We showed in functional studies that MARCO not only binds heat-killed bacteria, but also living bacteria. We also demonstrated that LPS itself is recognized by MARCO. Moreover, we have produced and purified recombinant domain V, and studied whether a surface coated with this protein supports binding of bacteria. This study indicated that compared to soluble MARCO, the monomeric domain V has low, barely detectable bacteria-binding activity. This finding highlights the importance of trimerization for the bacteria-binding activity of MARCO. In the fourth part of this work, we show a unique role for the marginal zone macrophages in the controlling of the retention and trafficking of the marginal zone B cells. This work originated from the observation that in mice deficient in the inhibitory signaling molecule SH2-containing inositol-5-phosphatase 1, the marginal zone macrophages had migrated to the red pulp and the marginal zone B cells had disappeared. Experiments utilizing soluble MARCO demonstrated that the interaction between the marginal zone macrophages and the marginal zone B cells is mediated by MARCO on the macrophages and its unknown ligand on the B cells. In the last part of this study, we first show using plasmon surface resonance technique that soluble MARCO binds LPS, LTA and poly(I). We detected no binding to heparin which is in line with the fact that heparin is not a ligand of scavenger receptors although it is a negatively charged macromolecule. Attempting to find novel ligands of MARCO, we then utilized the phage display technology, and searched for MARCO-binding phages from a random, complex decapeptide phage library. The screening resulted in selection of novel MARCO-binding peptides. Interestingly, the peptides were found to bind to the SRCR domain of MARCO. This finding strengthens the notion that the SRCR domains constitute the biologically active part in MARCO. Current studies are directed towards finding the protein(s) that the selected peptides