John G. Raynes

Serum amyloid A (SAA): an acute phase protein and apolipoprotein.

Serum amyloid A (SAA) proteins comprise a family of apolipoproteins coded for by at least three genes with allelic variation and a high degree of homology between species. The synthesis of certain members of the family is greatly increased in inflammation. However, SAA is not often used as an acute-phase marker despite being at least as sensitive as C-reactive protein. SAA proteins can be considered as apolipoproteins since they associate with plasma lipoproteins mainly within the high density range, perhaps through amphipathic alpha-helical structure. It is not known why certain subjects expressing SAA develop secondary systemic amyloidosis. There is still no specific function attributed to SAA; however, a popular hypothesis suggests that SAA may modulate metabolism of high density lipoproteins (HDL). This may impede the protective function of HDL against the development of atherosclerosis. The potential significance of the association between SAA and lipoproteins needs further evaluation.

Serum Amyloid A Protein Binds to Outer Membrane Protein A of Gram-negative Bacteria

Serum amyloid A (SAA) is the major acute phase protein in man and most mammals. We observed SAA binding to a surprisingly large number of Gram-negative bacteria, including Escherichia coli, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. The binding was found to be high affinity and rapid. Importantly, this binding was not inhibited by high density lipoprotein with which SAA is normally complexed in serum. Binding was also observed when bacteria were offered serum containing SAA. Ligand blots following SDS-PAGE or two-dimensional gels revealed two major ligands of 29 and 35 kDa that bound SAA when probing with radiolabeled SAA or SAA and monoclonal anti-SAA. Following fractionation the ligand was found in the outer membrane fraction of E. coli and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be outer membrane protein A (OmpA). OmpA-deficient E. coli did not bind SAA, and following purification of OmpA the protein retained binding activity. The ligands on other bacteria were likely to be homologues of OmpA because wild type, but not OprF-deficient, P. aeruginosa bound SAA.

Proteolysis of AA Amyloid Fibril Proteins by Matrix Metalloproteinases-1, -2, and -3

We recently demonstrated the presence of matrix metalloproteinases (MMPs)-1, -2, and -3 in AA amyloid deposits, which lead us to speculate that MMPs may participate in amyloidogenesis by either processing the precursor protein, or by degrading the amyloid deposits. Here we investigated this theory by determining the ability of MMP-1, -2, and -3 to degrade human acute-phase serum amyloid A (SAA) and human AA amyloid fibril proteins (AFPs). The following in vitro degradation experiments were performed: using either recombinant MMP-1, -2, or -3 and SAA as a substrate; using either recombinant MMP-1, -2, or -3 and AFP as a substrate; and using THP-1 cells as the protease source and AFP as the substrate. All three MMPs were able to cleave SAA and AFP within the region spanning residues 51 to 57. The following cleavage sites were identified: at 57 to 58 for MMP-1; at 7 to 8 and 51 to 52 for MMP-2; at 7 to 8, 16 to 17, 23 to 24, 51 to 52, 55 to 56, 56 to 57, and 57 to 58 for MMP-3. Cell culture experiments showed that THP-1 cells were able to degrade AFPs. Degradation was significantly delayed after addition of a general metalloproteinase inhibitor (o-phenanthroline) to dextran sulfate-stimulated cells. This is the first study to show that human SAAs and AFPs are susceptible to proteolytic cleavage by MMPs. Immunocytochemistry and electron microscopy showed that degradation takes place in the pericellular or extracellular compartment.

CD11b Regulates Recruitment of Alveolar Macrophages but Not Pulmonary Dendritic Cells after Pneumococcal Challenge

Despite their close physical and functional relationships, alveolar macrophages (AMs) and pulmonary dendritic cells (pulDCs) have rarely been examined together in the context of infection. Using a nonlethal, resolving model of pneumonia caused by intranasal injection of Streptococcus pneumoniae, we demonstrate that AMs and pulDCs exhibit distinct characteristics during pulmonary inflammation. Recruitment of AMs and pulDCs occurred with different kinetics, and increased numbers of AMs resulted mainly from the appearance of a distinct subset of CD11b(High) AMs. Increased numbers of CD11b(High) and CD11b(Low) AMs, but not pulDCs, were recoverable from bronchoalveolar lavage fluid. CD11b expression on AMs was significantly increased by granulocyte-macrophage colony-stimulating factor but not by interleukin-10 or pathogen-associated stimuli. Finally, antibody blockade demonstrated that CD11b was critical for the recruitment of AMs, but not pulDCs, into the lung after pneumococcal challenge. These data demonstrate that there are significant differences between AM and pulDC responses to inflammatory pathogenic stimuli in vivo.

Specific Interactions Between Sense and Complementary Peptides: The Basis for the Proteomic Code

After the publication of the review, the authors would also like to offer the following provisional definition of the proteomic code as an aid to discussion: The proteomic code can be provisionally defined as strategic pairs of amino acid residues that make specific contact/interactions with each other through space. These strategic pairs may correspond to M-I pairs in the first instance (specified by the genetic code and its complement) or to M-I pair derivatives thereof. Table 3. Table to show how the Root-Bernstein (R-B) pairs of amino acid residues are derived.

C‐reactive protein‐mediated phagocytosis and phospholipase D signalling through the high‐affinity receptor for immunoglobulin G (FcγRI)

C‐reactive protein (CRP) is the prototypic acute‐phase protein in man which performs innate immune functions. CRP‐mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcγRs) or even a putative CRP‐specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcγRI with a threefold higher affinity than IgG (KD = 0·81 × 10−9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC‐conjugated sheep erythrocytes increased phagocytosis of these particles by COS‐7 cells transfected with FcγRI‐II chimaera or FcγRI/γ‐chain. Interferon‐γ‐treated U937 cells, which signal through FcγRI to activate phospholipase D (PLD) in response to cross‐linked IgG, were also activated by CRP without any requirement for further cross‐linking. These studies indicate that CRP is capable of binding to and cross‐linking FcγRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcγRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

THE ACUTE PHASE RESPONSE

1 Introduction 2 Induction of the Acute Phase Response 3 Acute Phase Proteins 4 Hemopoietic Responses 5 Metabolic Changes 6 Hormonal Changes 7 Fever and Other Behavioral Changes 8 Control of Systemic Inflammation 9 Disorders of the Acute Phase Response Keywords: acute phase response (APR), conserved response; ‘systemic inflammatory response syndrome (SIRS)’; liver protective responses; acute phase response induction; proinflammatory cytokines and acute phase proteins; acute phase proteins; C-reactive protein and other pentraxins; lipopolysaccharide-binding protein (LBP)

Acute-phase HDL in phospholipid transfer protein (PLTP)-mediated HDL conversion

In reverse cholesterol transport, plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and prebeta-HDL. During the acute-phase reaction (APR), serum amyloid A (SAA) becomes the predominant apolipoprotein on HDL. Displacement of apo A-I by SAA and subsequent remodeling of HDL during the APR impairs cholesterol efflux from peripheral tissues, and might thereby change substrate properties of HDL for lipid transfer proteins. Therefore, the aim of this work was to study the properties of SAA-containing HDL in PLTP-mediated conversion. Enrichment of HDL by SAA was performed in vitro and in vivo and the SAA content in HDL varied between 32 and 58 mass%. These HDLs were incubated with PLTP, and the conversion products were analyzed for their size, composition, mobility in agarose gels, and apo A-I degradation. Despite decreased apo A-I concentrations, PLTP facilitated the conversion of acute-phase HDL (AP-HDL) more effectively than the conversion of native HDL(3), and large fusion particles with diameters of 10.5, 12.0, and 13.8 nm were generated. The ability of PLTP to release prebeta from AP-HDL was more profound than from native HDL(3). Prebeta-HDL formed contained fragmented apo A-I with a molecular mass of about 23 kDa. The present findings suggest that PLTP-mediated conversion of AP-HDL is not impaired, indicating that the production of prebeta-HDL is functional during the ARP. However, PLTP-mediated in vitro degradation of apo A-I in AP-HDL was more effective than that of native HDL, which may be associated with a faster catabolism of inflammatory HDL.

The lipidation status of acute-phase protein serum amyloid A determines cholesterol mobilization via scavenger receptor class B, type I

During the acute-phase reaction, SAA (serum amyloid A) replaces apoA-I (apolipoprotein A-I) as the major HDL (high-density lipoprotein)-associated apolipoprotein. A remarkable portion of SAA exists in a lipid-free/lipid-poor form and promotes ABCA1 (ATP-binding cassette transporter A1)-dependent cellular cholesterol efflux. In contrast with lipid-free apoA-I and apoE, lipid-free SAA was recently reported to mobilize SR-BI (scavenger receptor class B, type I)-dependent cellular cholesterol efflux [Van der Westhuyzen, Cai, de Beer and de Beer (2005) J. Biol. Chem. 280, 35890-35895]. This unique property could strongly affect cellular cholesterol mobilization during inflammation. However, in the present study, we show that overexpression of SR-BI in HEK-293 cells (human embryonic kidney cells) (devoid of ABCA1) failed to mobilize cholesterol to lipid-free or lipid-poor SAA. Only reconstituted vesicles containing phospholipids and SAA promoted SR-BI-mediated cholesterol efflux. Cholesterol efflux from HEK-293 and HEK-293[SR-BI] cells to lipid-free and lipid-poor SAA was minimal, while efficient efflux was observed from fibroblasts and CHO cells (Chinese-hamster ovary cells) both expressing functional ABCA1. Overexpression of SR-BI in CHO cells strongly attenuated cholesterol efflux to lipid-free SAA even in the presence of an SR-BI-blocking IgG. This implies that SR-BI attenuates ABCA1-mediated cholesterol efflux in a way that is not dependent on SR-BI-mediated re-uptake of cholesterol. The present in vitro experiments demonstrate that the lipidation status of SAA is a critical factor governing cholesterol acceptor properties of this amphipathic apolipoprotein. In addition, we demonstrate that SAA mediates cellular cholesterol efflux via the ABCA1 and/or SR-BI pathway in a similar way to apoA-I.

FcγRIIa expression with FcγRI results in C-reactive protein- and IgG-mediated phagocytosis

C‐reactive protein (CRP) is a pattern‐recognition molecule, which can bind to phosphorylcholine and certain phosphorylated carbohydrates found on the surface of a number of microorganisms. CRP has been shown recently to bind human Fc receptor for immunoglobulin G (IgG; FcγR)I and mediate phagocytosis and signaling through the γ‐chain. To date, binding of monomeric CRP to FcγRII has been contentious. We demonstrate that erythrocytes opsonized with CRP bind FcγRIIa‐transfected COS‐7 cells. In addition, we demonstrate that FcγRI can use FcγRIIa R131 and H131 to phagocytose erythrocytes coated with IgG or purified or recombinant CRP in the absence of the γ‐chain. COS‐7 cells expressing FcγRIIa or FcγRI alone did not phagocytose opsonized erythrocytes. Such phagocytosis required the cytoplasmic domain of FcγRIIa, as mutation of tyrosine at position 205 and truncation of the cytoplasmic domain from the end of the transmembrane region (position 206), resulting in the loss of the immunoreceptor tyrosine activatory motif, abrogated phagocytosis. FcγRIIa R131 was more efficient than FcγRIIa H131 at mediating CRP‐dependent phagocytosis.