Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

Serum amyloid A, an acute-phase protein, modulates proteoglycan synthesis in cultured murine peritoneal macrophages.

Cultured peritoneal macrophages obtained from azocasein-injected mice were found to produce several fold more cell-associated and medium proteoglycans than peritoneal macrophages from untreated mice. Since serum amyloid A (an acute-phase protein) is also upregulated following injections of azocasein, we questioned whether its production was the immediate agent stimulating proteoglycan formation. Cultured peritoneal macrophages from untreated mice were then incubated with varying concentrations of SAA, resulting in a similar dose-dependent several fold increase in proteoglycan production. Of particular note was a disproportionate increase in cell-associated heparan sulfate proteoglycans in both experimental groups and of dermatan sulfate and chondroitin sulfate proteoglycans when cells were incubated in the presence of SAA in the culture medium. These results indicate a potentially important function of SAA in directing specific modifications in inflammatory conditions where increase in macrophage proteoglycans may play direct roles.     

Immunolocalization of lipid peroxidation/advanced glycation end products in amyloid A amyloidosis

Chronic inflammation, superimposed by amyloid fibril deposition, is believed to trigger the cascade of oxidative stress response in the affected organs and tissues. We examined immunohistochemically the distribution of 4-hydroxy-2-nonenal (HNE) and N(epsilon)-(carboxymethyl)lysine (CML), markers of lipid peroxidation and advance glycation end products (AGE), respectively, in spleen sections and peritoneal macrophages (MPhi) from mice before and during AA amyloidosis. With time, both HNE and CML immunoreactivities increased significantly in MPhi and splenic reticuloendothelial cells, known to be associated with the clearance of serum amyloid A, the precursor of AA fibrils. HNE and CML were localized to the plasma membrane and the cytoplasmic compartment of MPhi and HNE only at the nuclear membrane. These markers were also colocalized bound to AA fibrils infiltrating the splenic sinus walls. Our results reinforce the notion that oxidative stress is an integral component of amyloidotic tissues. Both lipid peroxidation and AGE have been implicated in protein modification and amyloid fibril formation. The significance of HNE and CML associated with the monocytoid cells and implicated in SAA clearance and AA fibril formation, is discussed with the pathogenesis of AA fibrils.     

The acute-phase proteins serum amyloid A and C reactive protein in transudates and exudates

The distinction between exudates and transudates is very important in the patient management. Here we evaluate whether the acute-phase protein serum amyloid A (SAA), in comparison with C reactive protein (CRP) and total protein (TP), can be useful in this discrimination. CRP, SAA, and TP were determined in 36 exudate samples (27 pleural and 9 ascitic) and in 12 transudates (9 pleural and 3 ascitic). CRP, SAA, and TP were measured. SAA present in the exudate corresponded to 10% of the amount found in serum, that is, the exudate/serum ratio (E/S) was 0.10 +/- 0.13. For comparison, the exudate/serum ratio for CRP and TP was 0.39 +/- 0.37 and 0.68 +/- 0.15, respectively. There was a strong positive correlation between serum and exudate SAA concentration (r = 0.764; p < 0.0001). The concentration of SAA in transudates was low and did not overlap with that found in exudates (0.02-0.21 versus 0.8-360.5 g/mL). SAA in pleural and ascitic exudates results mainly from leakage of the serum protein via the inflamed membrane. A comparison of the E/S ratio of SAA and CRP points SAA as a very good marker in discriminating between exudates and transudates.     

Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1β is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1β production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro-IL-1β expression and for inflammasome activation, resulting in secretion of mature IL-1β. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X(7) abrogated the release of mature IL-1β. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1β secretion was markedly reduced in ASC(-/-) macrophages, and silencing NLRP3 decreased IL-1β secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro-IL-1β and activation of the NLRP3 inflammasome via P2X(7) receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1β in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.     

Serum amyloid A protects murine macrophages from lethal toxin-mediated death

Lethal toxin, a key virulence factor produced by Bacillus anthracis, induces cell death, in part by disrupting numerous signaling pathways, in mouse macrophages. However, exposure to sublethal doses of lethal toxin allows some cells to survive. Because these pro-survival signaling events occur within a few hours after exposure to sublethal doses, we hypothesized that acute phase proteins might influence macrophage survival. Our data show that serum amyloid A (SAA) is produced in response to lethal toxin treatment. Moreover, pre-treatment of macrophages with exogenous SAA protected macrophages from lethal toxin-mediated death. Exogenous SAA activated the p38 mitogen activated protein kinase (MAP) kinase pathway, while lethal toxin mutants incapable of p38 activation were incapable of causing cell death. Chemical inhibition of the p38 activation pathway abrogated the protective effects of SAA. These data show that SAA affords protection against lethal toxin in mouse macrophages and link this response to the p38 pathway.     

Acute phase induction of mouse serum amyloid P component. Correlation with other parameters of inflammation

Hepatic mRNA levels of the mouse major acute phase proteins serum amyloid P component (SAP) and serum amyloid A component (SAA) were monitored at timed intervals after i.p. injection of thioglycollate or s.c. injection of azocasein. Both mRNA increased dramatically in response to either inflammatory stimulus. The increase in SAA mRNA levels accompanied an abrupt change in mRNA size from 650 to 750 bases. Peak SAA mRNA concentrations were observed 18 h after either stimulus; by 72 h concentrations had returned to preinflammatory levels. Peak SAP mRNA concentrations were observed 8 h after thioglycollate and 12 to 18 h after azocasein injection; by 36 h concentrations were close to preinflammatory levels. All mRNA species studied (SAP, SAA and the complement components C3, C5 and factor B) were induced more rapidly by the thioglycollate stimulus and reached higher peak concentrations. SAP mRNA levels were correlated with other parameters of inflammation: infiltration of peritoneal exudate cells (PEC) into the peritoneum after thioglycollate injection, and serum concentrations of SAP after azocasein injection. Serum SAP concentrations rose 20-fold in response to the latter stimulus by 36 h, i.e., 18 to 24 h after the peak SAP mRNA levels. The highest numbers of PEC were present 24 h after the thioglycollate stimulus, i.e. 16 h after the maximum SAP mRNA concentration, indicating the continuation of an active local inflammation many hours after one aspect of the systemic response has ceased.     

Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice

Although serum amyloid A (SAA) is an excellent marker for coronary artery disease, its direct effect on atherogenesis in vivo is obscure. In this study we investigated the direct effect of SAA on promoting the formation of atherosclerosis in apolipoprotein E-deficient (ApoE?/?) mice. Murine SAA lentivirus was constructed and injected into ApoE?/? mice intravenously. Then, experimental mice were fed a chow diet (5% fat and no added cholesterol) for 14 wks. The aortic atherosclerotic lesion area was larger with than without SAA treatment. With increased SAA levels, the plasma levels of interleukin-6 and tumor necrosis factor-α were significantly increased. Macrophage infiltration in atherosclerotic regions was enhanced with SAA treatment. A migration assay revealed prominent dose-dependent chemotaxis of SAA to macrophages. Furthermore, the expression of monocyte chemotactic protein-1 and vascular cell adhesion molecule-1 (VCAM-1) was upregulated significantly with SAA treatment. SAA-induced VCAM-1 production was detected in human aortic endothelial cells in vitro. Thus, an increase in plasma SAA directly accelerates the progression of atherosclerosis in ApoE?/? mice. SAA is not only a risk marker for atherosclerosis but also an active participant in atherogenesis.     

Echinococcus multilocularis: relationship between persistent inflammation, serum amyloid A protein response and amyloidosis in four mouse strains

LPS-hyporesponsive (C3H/HeJ) and LPS-sensitive (C57BL/6, CBA/J, C3H/HeSn) strains of mice were infected intraperitoneally with 50 alveolar hydatid cysts (AHC) to assess the effect of protracted severe inflammation on serum amyloid A protein (SAA) concentrations, splenic amyloid deposition, and pre- and postamyloidotic alterations in the splenic architecture. In general, the SAA concentrations in all the four mouse strains showed a moderate but steady increase throughout the course of infection. Splenic amyloid deposition commenced between 6 to 8 weeks postinfection (p.i.) when the SAA concentrations were relatively low and increased progressively until 12 weeks p.i. when 52 to 78% of the splenic parenchyma was obliterated. CBA mice which harbored the largest AHC throughout the 12-week course of infection showed the poorest SAA and amyloid responses; the situation was reversed in the C3H/HeSn strain. Histologically, most of the splenic follicles, during the stage of maximum amyloid deposition, appeared hypocellular. Their T-cell-dependent periarterial sinuses were either totally depleted of cells or contained plasma cells or myeloid cells. These results show that (a) there is no direct correlation between the intensity of inflammation, SAA concentrations, or amounts of amyloid deposition in either of the four mouse strains and (b) amyloidosis secondary to AHC infection differs from other experimental mouse models of amyloidosis in the magnitude of SAA elevation during the preamyloid phase.     

Dietary fish oil modulation of macrophage amyloid P component responses in mice

Arthritis-susceptible B10.RIII mice, maintained on either fish oil (FO) or corn oil (CO) diets (5% by weight), and amyloid-susceptible CBA/J mice fed chow diets were given 20 micrograms purified LPS by i.p. injection. Both strains of mice responded to LPS with a 20- to 30-fold increase in plasma amyloid P component (AP) levels. There were no differences in the response between males and females or between FO and CO treatment groups. The data demonstrated that cultured peritoneal macrophages (M phi) respond to LPS stimulation with increased secretion of AP. In contrast to plasma AP levels, the MO response to LPS stimulation, as measured by production of AP, was influenced by both gender and diet. Although M phi from both male and female mice on the CO diet and male mice on the FO diet responded similarly, those from female mice on the FO diet secreted only 25 to 35% as much AP as did the other three groups. There were no dietary effects on the LPS-induced serum amyloid A protein response nor was there detectable serum amyloid A protein produced by the M phi. These results demonstrate that unstimulated, resident peritoneal M phi secrete AP as a normal constituent and in increasing amounts in response to LPS stimulation.     

Cellular mechanism of fibril formation from serum amyloid A1 protein

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high‐density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.     

Macrophage cholesterol efflux and the active domains of serum amyloid A 2.1

Serum amyloid A 2.1 (SAA2.1) suppresses ACAT and stimulates cholesteryl ester hydrolase (CEH) activities in cholesterol-laden macrophages, and in the presence of a cholesterol transporter and an extracellular acceptor, there is a marked increase in the rate of cholesterol export in culture and in vivo. The stimulation of CEH activity by SAA2.1 is not affected by chloroquine, suggesting that it operates on neutral CEH rather than the lysosomal form. With liposomes containing individual peptides of SAA2.1, residues 1-20 inhibit ACAT activity, residues 74-103 stimulate CEH activity, and each of residues 1-20 and 74-103 promotes macrophage cholesterol efflux to HDL in culture media. In combination, these peptides exhibit a profound effect, so that 55-70% of cholesterol is exported to media HDL in 24 h. The effect is also demonstrable in vivo. [3H]cholesterol-laden macrophages injected intravenously into mice were allowed to establish themselves for 24 h. Thereafter, the mice received a single intravenous injection of liposomes containing intact SAA1.1, SAA2.1, peptides composed of SAA2.1 residues 1-20, 21-50, 51-80, 74-103, or SAA1.1 residues 1-20. Only liposomes containing intact SAA2.1 or its residues 1-20 or 74-103 promoted the efflux of cholesterol in vivo. A single injection of each of the active peptides is effective in promoting cholesterol efflux in vivo for at least 4 days.     

Serum amyloid A, cytokines, and corticosterone responses in germfree and conventional mice after lipopolysaccharide injection.

To determine why germfree mice are less susceptible to lipopolysaccharide (LPS) than conventional mice, we studied serum levels of serum amyloid A (SAA), tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, and corticosterone in mice after treatment with LPS. A single injection of LPS caused an elevation of SAA, an acute-phase protein in the mouse, in both conventional and germfree IQI mice, and the response was significantly less in germfree mice. LPS-induced elevations of serum TNF, IL-1, and IL-6 levels were also significantly less in germfree mice, while serum corticosterone levels were greater in germfree mice than in conventional mice. These results suggest that the lower susceptibility to LPS and a smaller response of SAA elevation by LPS in germfree mice may result from less elevation in serum of these cytokines in these mice, which are known to mediate the acute phase response of SAA. High levels of serum corticosterone in germfree mice may be partly responsible for the lower responsiveness of these inflammatory cytokines to LPS in these mice.     

Increased serum amyloid a levels reflect colitis severity and precede amyloid formation in IL-2 knockout mice

The lack of sensitive and relatively non-invasive measures has hampered monitoring the clinical course of spontaneously developing colitis in IL-2-deficient (-/-) mice. We selected (i) to study the correlation of the acute phase plasma proteins serum amyloid A (SAA) and serum amyloid P component (SAP) levels with colonic disease and (ii) to characterize the amyloidosis in the IL-2(-/-)animals. IL-2(-/-)mice exhibited increasing severity of gross intestinal inflammation with age, confined to the distal colon. Histologically, the colonic disease score increased serially in IL-2(-/-)animals. Wild-type mice showed no activity, while 16-week-old IL-2(+/-)animals had minimal colitis with small ulcers and lamina propria inflammatory infiltrate. Periportal hepatitis was present and positive Congo red staining indicated amyloidosis of the liver and spleen in 16 week IL-2(-/-)mice. SAA immunostaining in the liver and spleen was increased in the 8 week and 16 week IL-2(-/-)and 16 week IL-2(+/-)animals indicating AA amyloid deposits. Plasma SAA and SAP levels were markedly elevated, and generally preceded the onset of colitis and reflected its severity. Northern analysis showed markedly increased SAA expression in the liver and intestine of IL-2(-/-)and intestine of IL-2(+/-)16-week-old animals. Increased intestinal expression of SAA3 (lamina propria macrophages) indicates local inflammation in IL-2(+/-)animals at 16 weeks. Treatment of 3-week-old animals with systemic IL-2 or IL-1 receptor antagonist (IL-1ra) delayed inflammation, postponed the increase in SAA levels and minimized disease onset. These results further demonstrate that IL-2 plays a significant role in normal immune responses in the body and that plasma SAA levels both reflect colonic disease severity and may indicate subclinical disease in both IL-2(-/-)and IL-2(+/-)mice. Furthermore. The mechanism of IL-2-deficient induced colitis appears to be mediated in part through the increase in IL-1. In addition, the IL-2(-/-)mouse of spontaneous enterocolitis may provide a unique system for studying spontaneously developing AA amyloidosis.     

Cutting edge: TLR2 is a functional receptor for acute-phase serum amyloid A

Induced secretion of acute-phase serum amyloid A (SAA) is a host response to danger signals and a clinical indication of inflammation. The biological functions of SAA in inflammation have not been fully defined, although recent reports indicate that SAA induces proinflammatory cytokine expression. We now show that TLR2 is a functional receptor for SAA. HeLa cells expressing TLR2 responded to SAA with potent activation of NF-kappaB, which was enhanced by TLR1 expression and blocked by the Toll/IL-1 receptor/resistance (TIR) deletion mutants of TLR1, TLR2, and TLR6. SAA stimulation led to increased phosphorylation of MAPKs and accelerated IkappaBalpha degradation in TLR2-HeLa cells, and results from a solid-phase binding assay showed SAA interaction with the ectodomain of TLR2. Selective reduction of SAA-induced gene expression was observed in tlr2-/- mouse macrophages compared with wild-type cells. These results suggest a potential role for SAA in inflammatory diseases through activation of TLR2.     

Extrahepatic production of acute phase serum amyloid A

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.     

Accelerated resolution of AA amyloid in heparanase knockout mice is associated with matrix metalloproteases

AA-amyloidosis is a disease characterized by abnormal deposition of serum A amyloid (SAA) peptide along with other components in various organs. The disease is a complication of inflammatory conditions that cause persistent high levels of the acute phase reactant SAA in plasma. In experimental animal models, the deposited amyloid is resolved when the inflammation is stopped, suggesting that there is an efficient clearance mechanism for the amyloid. As heparan sulfate (HS) is one of the major components in the amyloid, its metabolism is expected to affect the pathology of AA amyloidosis. In this study, we investigated the effect of heparanase, a HS degradation enzyme, in resolution of the AA amyloid. The transgenic mice deficient in heparanase (Hpa-KO) produced a similar level of SAA in plasma as the wildtype control (Ctr) mice upon induction by injection of AEF (amyloid enhancing factor) and inflammatory stimuli. The induction resulted in formation of SAA amyloid 7-days post treatment in the spleen that displayed a comparable degree of amyloid load in both groups. The amyloid became significantly less in the Hpa-KO spleen than in the Ctr spleen 10-days post treatment, and was completely resolved in the Hpa-KO spleen on day 21 post induction, while a substantial amount was still detected in the Ctr spleen. The rapid clearance of the amyloid in the Hpa-KO mice can be ascribed to upregulated matrix metalloproteases (MMPs) that are believed to contribute to degradation of the protein components in the AA amyloid. The results indicate that both heparanase and MMPs play important parts in the pathological process of AA amyloidosis.     

Serum amyloid A promotes LPS clearance and suppresses LPS‐induced inflammation and tissue injury

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram‐negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute‐phase proteins, but the relationship between SAA expression and LPS‐induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα‐induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1‐LPS interaction with a SAA1‐derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute‐phase SAA provides innate feedback protection against LPS‐induced inflammation and tissue injury.     

Polymorphism of tissue and serum amyloid A (AA and SAA) proteins in the mouse

Amino acid sequence studies of the amino terminal 25 residues of amyloid A (AA) protein and the serum precursor (SAA) induced with casein or LPS indicate differences in the sequence at position 6 and significant heterogeneity at several other positions in SAA. These findings suggest that SAA is a polymorphic serum protein and raise the possibility that only certain forms of SAA are processed to the tissue amyloid fibril.     

Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences

The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.