Developmental role and ligand binding properties of macrophage scavenger receptor MARCO

Macrophages express several cell surface receptors, that mediate the recognition of a wide range of endogenous and exogenous ligands. In this manner, macrophages are able to perform their multiple biological functions in regulation of homeostasis and host defense. These surface receptors recognize ligands, either indirectly by an opsonization mechanism, or directly by pattern recognition.

Pattern recognition receptors (PRR) are important in the initiation of innate immune response. Scavenger receptors (SRs) are PRRs with a common specificity of binding to polyanionic ligands. MARCO (MAcrophage Receptor with a COllagenous structure) and SR-A (Scavenger Receptor A), that belong to class A SRs, share domain structural properties and ligand repertoire, but they differ with respect to tissue distribution and ligand binding properties.

In contrast to the broad expression pattern of SR-A, expression of MARCO is restricted to a subset of tissue macrophages, i.e. macrophages in the spleen marginal zone (MZ), lymph nodes, and resident macrophages of the peritoneal cavity. The strategic positioning of MARCO on the spleen MZ macrophages suggests a role of MARCO in the removal of circulating organisms, as well as in the initiation of TI-2 (Thymus -Independent type 2) response, which depends on an intact spleen MZ. To study the in vivo biological role of MARCO, we have generated MARCOdeficient and MARCO/SRA-double-deficient mice. These mice appear normal in a germ-free environment. However, the development of the spleen MZ is defective in these mice. Knockout mice showed delayed development of the MZ during ontogeny, and its microarchitecture was still immature at adult age.

During ontogeny, MARCO-positive cells are spread throughout the entire spleen at the day of birth, but they form the MZ structure in the following week. Following the formation of the MARCOpositive MZ, the macrophages started to develop and differentiate to express other receptors, such as SIGNR-1 and Siglec-1. A similar finding was observed during reappearence of spleen macrophages after liposome-induced spleen injury in adult mice. The defective microarchitecture of the spleen MZ led to an impaired TI-2 response in knockout mice, which was observed when mice were challenged with a pneumococcal polysaccharide vaccine, a TI-2 antigen. Besides the defects in the spleen MZ, the ablation of MARCO results in a significant reduction in the size of resident peritoneal macrophage population. Since SR-A is also strongly expressed on macrophages in the spleen MZ and peritoneal cavity, all the above-described phenotypes were, not surprisingly, more striking in mice lacking both MARCO and SR-A receptors. These findings suggest that MARCO and SR-A, in addition to being bacteria-binding receptors, possibly interact with endogenous ligands through which they regulate the positioning and differentiation of macrophages in vivo.

Identification of ligands is an important aspect of PRR research. We have taken the advantage of an unbiased approach, phage display, to search for novel MARCO ligands using soluble recombinant MARCO (sMARCO) protein. This resulted in the enrichment of several hydrophobic peptides, which was contrary to the previous understanding that the ligands of scavenger receptors are polyanionic. BlAcore analysis confirmed that the hydrophobic peptides are ligands for sMARCO, and have a higher affinity than the known MARCO ligands LPS and LTA. Database search suggested that the most enriched peptide sequence represents complement C4, but so far we have not got convincing experimental results to support this suggestion. Further work with the two most enriched peptides demonstrated that these peptides bind to the SRCR domain of the MARCO molecule. A study with chimeric scavenger receptors indicated that even minor sequence changes in the SRCR domain can have profound effects on the binding of the prototypic scavenger receptor ligand, AcLDL This study strengthens the notion that the SRCR domain is the major ligand-binding domain in MARCO, and that this domain contains multiple ligand-binding sites.

The results of the last part of my thesis work demonstrate that MARCO recognizes Neisseria meningitidis (NM), an important human pathogen. Interestingly, we found that MARCO binds both wild-type NM as well as a mutant strain which lacks lipid A, indicating that LPS and LTA are not the only bacterial ligands of MARCO. However, although the studies with the peritoneal macrophages from different knockout strains indicate that both MARCO and SR-A participitate in the binding/phagocytosis of NM, neither receptor is required for NM-stimulated TNF-á and nitric oxide production. Thus, the results suggest that TLR-dependent induction of MARCO during infection plays primarily a role in the clearance of invading pathogens.

List of scientific papers

I. Chen Y, Pikkarainen T, Elomaa O, Soininen R, Kodama T, Kraal G, Tryggvason K (2005). Defective microarchitecture of the spleen marginal zone and impaired response to a thymus-independent type 2 antigen in mice lacking scavenger receptors MARCO and SR-A. J Immunol. 175(12): 8173-80.
https://pubmed.ncbi.nlm.nih.gov/16339556

II. Chen Y, Sankala M, Ojala JR, Sun Y, Tuuttila A, Isenman DE, Tryggvason K, Pikkarainen T (2006). A phage display screen and binding studies with acetylated low density lipoprotein provide evidence for the importance of the scavenger receptor cysteine-rich (SRCR) domain in the ligand-binding function of MARCO. J Biol Chem. 281(18): 12767-75.
https://pubmed.ncbi.nlm.nih.gov/16524885

III. Mukhopadhyay S, Chen Y, Sankala M, Peiser L, Pikkarainen T, Kraal G, Tryggvason K, Gordon S (2006). MARCO, an innate activation marker of macrophages, is a class A scavenger receptor for Neisseria meningitidis. Eur J Immunol. 36(4): 940-9.
https://pubmed.ncbi.nlm.nih.gov/16525990