F. C. De Beer

Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures.

We previously reported that high density lipoprotein (HDL) protects against the oxidative modification of low density lipoprotein (LDL) induced by artery wall cells causing these cells to produce pro-inflammatory molecules. We also reported that enzyme systems associated with HDL were responsible for this anti-inflammatory property of HDL. We now report studies comparing HDL before and during an acute phase response (APR) in both humans and a croton oil rabbit model. In rabbits, from the onset of APR the protective effect of HDL progressively decreased and was completely lost by day three. As serum amyloid A (SAA) levels in acute phase HDL (AP-HDL) increased, apo A-I levels decreased 73%. Concomitantly, paraoxonase (PON) and platelet activating factor acetylhydrolase (PAF-AH) levels in HDL declined 71 and 90%, respectively, from days one to three. After day three, there was some recovery of the protective effect of HDL. AP-HDL from human patients and rabbits but not normal or control HDL (C-HDL) exhibited increases in ceruloplasmin (CP). This increase in CP was not seen in acute phase VLDL or LDL. C-HDL incubated with purified CP and re-isolated (CP-HDL), lost its ability to inhibit LDL oxidation. Northern blot analyses demonstrated enhanced expression of MCP-1 in coculture cells treated with AP-HDL and CP-HDL compared to C-HDL. Enrichment of human AP-HDL with purified PON or PAF-AH rendered AP-HDL protective against LDL modification. We conclude that under basal conditions HDL serves an anti-inflammatory role but during APR displacement and/or exchange of proteins associated with HDL results in a pro-inflammatory molecule.

Human serum amyloid A (SAA) protein: a prominent acute‐phase reactant for clinical practice

Serum amyloid A (SAA) proteins comprise a family of apolipoproteins synthesized in response to cytokines released by activated monocytes/macrophages. Acute‐phase protein concentrations have been advocated as objective biochemical indices of disease activity in a number of different inflammatory processes. Clinical studies in large groups of patients with a variety of disorders confirmed the rapid production and exceptionally wide dynamic range of the SAA response. It is as sensitive a marker for the acute‐phase as C‐reactive protein (CRP). Recent studies indicate that SAA is the most sensitive non‐invasive biochemical marker for allograft rejection. Further studies comparing the measurement of SAA to CRP could reveal other indications for its specific use. These studies are now more feasible given newer assays to measure this acute‐phase reactant. Observations that the acute‐phase response is tightly coupled to lipoprotein abnormalities and the fact that acute‐SAA proteins are mainly associated with plasma lipoproteins of the high density range suggested a possible role of this apolipoprotein (apo SAA) in the development of atherosclerosis. The expression of SAA mRNA in human atherosclerotic lesions and the induction of acute‐phase SAA by oxidized low‐density lipoproteins strengthen the hypothesis that SAA might play a role in vascular injury and atherogenesis.

Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides☆

We recently described 17 anti-CRP mAb, seven to native- (or conformational) and 10 to neo- (or sequence-determined) epitopes, including several anti-neo-CRP mAb specific for CRP peptide 199-206. In the present study, four new anti-native- and four new anti-neo-CRP mAb were generated and characterized by ELISA reactivity with native and modified human and rabbit CRP, as well as binding to pronase fragments of human CRP in Western blots. Assays with 17 synthetic CRP peptides identified anti-neo-CRP mAb specific for peptides 1-16, 14-24 and 137-152, respectively. The anti-neo-CRP mAb were reacted with fragments obtained by digesting CRP with multiple additional enzymes, including Staphylococcal V8 protease, trypsin, elastase, plasmin, thrombin and alpha-chymotrypsin. Native CRP was remarkably resistant to enzymic digestion, particularly in the presence of calcium, but was readily cleavable upon denaturation. Twenty-three informative fragments served to further distinguish mAb reactivity with at least four additional neo-CRP epitopes, which presumptively included residues in the regions of amino acids 22-45, 41-61, 114-121 and 130-138, respectively. The eight epitopes identified corresponded well with predicted regions of CRP antigenicity. In addition, at least six distinct native or conformation-determined epitopes were delineated. Reactivity of the anti-neo-CRP mAb with fragments of CRP generated by PMN enzymes indicated that regions sensitive to cleavage by neutrophil enzymes are located at approximately 3, 10 and 16 kD from the amino terminus of the CRP subunit. We expect that the anti-CRP mAb described and mapped herein will be useful tools for the elucidation of CRP structure and function.

Biosynthesis and Processing of SAA by Mouse L-Cells Transfected with the Human SAAg9 Gene

Pulse-chase studies of SAA biosynthesis and processing in mouse L-cells transfected with the SAAg9 gene have shown that, over a 2h time period, intracellular SAA does not show amino-terminal trimming. Extracellular SAA, although principally composed of the pI=8.0 isoform, shows limited conversion from the pI=8.0 to pl=7.4 isoform over a 2h period.

Identification of apo-SAA isoforms in man and mouse

Isoelectric identification of apo-SAA isoforms in mouse and human plasma is important in analyzing function and involvement in amyloidogenesis. In this paper we show the influence of altering the pH gradient on the migration of the human isoforms and identify novel minor apo-SAA isoforms. The NH2-terminals of these isoforms are analyzed by sequencing of the isoforms electroblotted onto polyvinylidene difluoride (PVDF) membranes. Integrity of the COOH-terminal is confirmed by blotting using a rabbit anti-human apo-SAA (aa 95-104) antibody. Isoelectric focusing (IEF) is compared to urea-SDS-acrylamide gel electrophoresis for the analysis of apo-SAA isoforms in haplotype A and B mouse strains [1,2].