ARTICLE |
Correspondence to: Simcha UrieliShoval, Hematology Unit, Hadassah U. Hospital, Mount Scopus, Jerusalem 91240, Israel..
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum amyloid A (SAA) is an acute-phase reactant whose level in the blood is elevated to 1000-fold as part of the body's responses to various injuries, including trauma, infection, inflammation, and neoplasia. As an acute-phase reactant, the liver has been considered to be the primary site of expression. However, limited extrahepatic SAA expression was described in mouse tissues and in cells of human atherosclerotic lesions. Here we describe nonradioactive in situ hybridization experiments revealing that the SAA mRNA is widely expressed in many histologically normal human tissues. Expression was localized predominantly to the epithelial components of a variety of tissues, including breast, stomach, small and large intestine, prostate, lung, pancreas, kidney, tonsil, thyroid, pituitary, placenta, skin epidermis, and brain neurons. Expression was also observed in lymphocytes, plasma cells, and endothelial cells. RT-PCR analysis of selected tissues revealed expression of the SAA1, SAA2, and SAA4 genes but not of SAA3, consistent with expression of these genes in the liver. Immunohistochemical staining revealed SAA protein expression that co-localized with SAA mRNA expression. These data indicate local production of the SAA proteins in histologically normal human extrahepatic tissues. (J Histochem Cytochem 46:13771384, 1998)
Key Words: SAA, human tissues, epithelium
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum amyloid A (SAA), a multigene family associated with high-density lipoproteins, is found in all mammals and birds examined thus far (
The properties and the physiological significance of the SAA family of proteins in normal and in disease states are poorly understood. Several functions have been proposed, including depression of the immune responses (
In the present study we describe nonradioactive in situ mRNA hybridization and immunohistochemistry experiments revealing SAA mRNA and protein expression in many histologically normal human extrahepatic tissues, predominantly in their epithelial components. In addition, RT-PCR analysis of these tissues revealed the expression of the SAA1, SAA2, and SAA4 genes but not of the SAA3 gene.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissues
Multisection slides containing a composite of histologically normal human tissue sections arranged on a grid (
Probes
SAA probe was prepared from pGEM transcription vector that contained a 110-BP sequence of mouse SAA1 cDNA (p125) (
In Situ Hybridization
In situ hybridization was performed as previously described (
Immunohistochemistry
Immunohistochemistry was performed using the Histostain- Plus SP kit, which offers superior sensitivity (Zymed Laboratories; San Francisco, CA). Deparaffinized and rehydrated sections were digested with protease (Sigma #P8038; St Louis, MO) (0.1% in PBS, 10 min, RT) and then treated according to the manufacturer's instructions with the ready-to-use kit components. The primary antibody used (at 1:20 dilution) was clone "mcl," which is a monoclonal antibody against amyloid fibril protein AA (
RT-PCR
Total RNA from fresh frozen tissues was prepared using Tri Reagent (Molecular Research Center; Cincinnati, OH). Synthesis of cDNA was performed using oligo(dT)1218 primer and the SuperScript II reverse transcriptase (Gibco BRL; Gaithersburg, MD). Amplification of cDNA was done using Red Hot DNA Polymerase (Advanced Biotechnologies; Surrey, UK) and primers specific for each of the four known human SAA genes; SAA1, 2, 3, and 4. The sequences of the various primers and their specificity were previously described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We applied the nonradioactive in situ hybridization technique, using digoxigenin-labeled SAA RNA probe (
|
We applied immunohistochemistry to determine whether the presence of SAA mRNA is accompanied by SAA protein synthesis. The antibody used was a monoclonal antibody against human amyloid fibril protein AA (
|
Breast
Strong in situ hybridization signal was observed in lobular epithelium and terminal duct epithelium; major duct epithelium stained weakly positive. Scattered lymphocytes and plasma cells stained positive. Surrounding loose and dense fibrous connective tissue stroma, fat tissue, and myoepithelial cells were negative (Figure 1A). Immunohistochemistry revealed SAA protein expression co-localized with SAA mRNA expression (Figure 2A). Figure 2B shows a negative control.
Large Intestine, Small Intestine, and Stomach
Positive in situ hybridization signal for SAA was observed in mucosal glandular epithelial cells. Lymphocytes and plasma cells scattered in the lamina propria and lymphocytes in lymphoid follicles of large intestine also stained positively. The smooth muscle of the muscularis mucosa and the loose fibrous connective tissue of the lamina propria and submucosa were negative (Figure 1B). Immunohistochemical staining revealed SAA protein expression, localized to the epithelium (Figure 2D).
Pancreas
Strong in situ hybridization signal was observed in exocrine glandular epithelium; the interlobular duct stained weakly positive. Interlobular septa and interstitial connective tissue were negative. Islets of Langerhans stained weakly positive (Figure 1C). Immunohistochemical staining revealed SAA protein expression localized to the epithelium (Figure 2C).
Prostate
Strong in situ hybridization signal was observed in glandular epithelium. The surrounding fibrous tissue stroma was negative, and scattered lymphocytes and plasma cells in the stroma stained positively (Figure 1D). Cystically dilated glands stained weakly positive. Immunohistochemical staining revealed SAA protein expression localized to the epithelium (Figure 2E). Figure 2F shows a negative control.
Lung
Strong in situ hybridization signal was observed in alveolar lining epithelium and in macrophages. In a medium-sized muscular artery, endothelial cells stained positively and muscle cells were negative. Interstitial connective tissue was negative (Figure 1E).
Skin
Strong in situ hybridization signal was observed in the epidermis (stratified squamous epithelium), whereas stratum corneum and dermis were negative (Figure 1F). In dermal blood vessels, focal positivity of endothelial cells was observed. The skin epidermis also stained positive by immunohistochemistry (not shown).
Brain
In situ hybridization signal was observed in the pyramidal neurons of the cerebral cortex and in Purkinje cells of the cerebellum, whereas non-neural support cells stained negatively (Figure 1G). Neurons also stained positively by immunohistochemistry (not shown).
Tonsil and Spleen
Positive in situ hybridization signal was observed in the crypt squamous epithelium of tonsil and in lymphocytes and plasma cells in the underlying stroma. Lymphocytes in lymphoid follicles of the tonsil stained strong positive (Figure 1H), as did the lymphocytes of the white pulp of the spleen (not shown).
In addition, positive staining was observed in the epithelial components of the following tissues: the distal and proximal convoluted tubules of the kidney, follicular epithelium of the thyroid, the secretory cells of the anterior pituitary, and the cytotrophoblast and syncytiotrophoblast lining the chorionic villi of placenta. Liver hepatocytes stained positively (not shown).
RT-PCR Analysis
We carried out RT-PCR analysis on RNA extracted from a few selected tissues to further support the in situ hybridization findings and to determine which of the four known human SAA genes are expressed. Oligonucleotide primers specific for each of the four known human SAA genes, SAA1, SAA2, SAA3, and SAA4, were used. This analysis revealed that fragments of the predicted sizes were amplified when the SAA1-, SAA2-, and SAA4-specific primers were used on RNA extracted from breast tissue. No PCR product was obtained with the SAA3-specific primers (Figure 3). PCR amplification products were sequenced and compared to the SAA sequences reported in the GenBank/EMBL Databases. The breast-derived SAA1 sequence had 99% nucleotide homology with liver-derived clones with accession numbers M10906 (clone pA1) and M23698 (
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum amyloid A is an acute-phase protein and has been considered to be expressed primarily in the liver. For many years, studies using animal or cultured cell models focused on investigating SAA expression and regulation in liver cells and on its possible function as a modulator of the acute-phase reaction and inflammation. Extrahepatic expression of the SAA genes was described, to a limited extent, in mouse tissues (
The role of SAA expressed in the epithelial components of human tissues is not clear. However, epithelium-specific gene expression is fundamental to both embryogenesis and the maintenance and function of adult tissues, and its impairment might be associated with diseases such as cancer. Our survey revealed that SAA is expressed in tissues of different epithelial types and degree of differentiation. Therefore, SAA may have a role in the maintenance of the epithelial phenotype similar to other epithelium-specific genes such as keratins or junction proteins (
The human SAA gene family is composed of four discrete loci containing two highly homologous genes, SAA1 and SAA2, and two less related genes, SAA3 and SAA4. SAA1 and SAA2 corresponding proteins are the predominant circulating SAA proteins during the acute-phase response (
The study was first done on commercial multitissue slides, enabling us to perform a large survey of tissues. Later, experiments were performed on histologically normal tissues from our archives (either fixedembedded tissues for in situ hybridization and immunohistochemistry or freshly frozen tissues for RT-PCR), revealing similar results. Furthermore, SAA mRNA and protein were also identified in tissues obtained from healthy individuals who underwent cosmetic procedures (breast, skin), suggesting epithelial SAA expression in the absence of a systemic acute-phase reaction. These results are in line with those of
In conclusion, the concept that SAA is expressed primarily in the liver can now be broadened because there is clearly SAA expression in human extrahepatic tissues. Therefore, in addition to SAA release to the circulation by the liver during an acute-phase response, one or more SAA proteins produced in human tissues may be constitutively expressed to serve as a first line of defense or in proper maintenance of human tissues (housekeeping role). The possibility that local SAA plays a role in the development of AA amyloidosis requires further investigation.
![]() |
Acknowledgments |
---|
Supported in part by the Israel Ministry of Absorption and the Israel Cancer Association.
We thank Dr R.L. Meek (the Heart Institute of Spokane) for the p125 cDNA clone, and Dr Y. Azar (Hadassah University Hospital) and Dr R.P. Linke (Max-Planck Institute) for helpful discussions.
Dedicated to the memory of the late Earl P. Benditt, MD.
Received for publication May 4, 1998; accepted August 18, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AldoBenson MA, Benson MD (1982) SAA suppression of immune response in vitro: evidence for an effect on T cell-macrophage interaction. J Immunol 128:2390-2392
Badolato R, Wang JM, Murphy WJ, Lloyd AR, Michiel DF, Bausserman LL, Kelvin DJ, Oppenheim JJ (1994) Serum amyloid A is a chemoattractant: induction of migration, adhesion and tissue infiltration of monocytes and polymorphonuclear leukocytes. J Exp Med 180:203-209[Abstract]
Battifora H, Mehta P (1990) The checkerboard tissue block: an improved multitissue control block. Lab Invest 63:722-724[Medline]
Baumberger C, Ulevitch RJ, Dayer JM (1991) Modulation of endotoxic activity of lipopolysaccharide by high-density lipoprotein. Pathobiology 59:378-383[Medline]
Betts JC, Edbrooke MR, Thakker RV, Woo P (1991) The human acute-phase serum amyloid A gene family: structure, evolution and expression in hepatoma cells. Scand J Immunol 34:471-482[Medline]
Birchmeier W, Behrens J (1994) Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Biochim Biophys Acta 1198:11-26[Medline]
Brinckerhoff CE, Mitchell TI, Karmilowicz MJ, KluveBeckerman B, Benson MD (1989) Autocrine induction of collagenase by serum amyloid A-like and ß2-microglobulin-like proteins. Science 243:655-657[Medline]
Eriksen N, Meek RL, Benditt EP (1993) The SAA lipoprotein family. In Mackiewicz A, Kushner I, Baumann H, eds. Acute Phase Proteins: Molecular Biology, Biochemistry and Clinical Applications. Boca Raton, FL, CRC Press, 93-106
Gatt ME, UrieliShoval S, PreciadoPatt L, Fridkin M, Calco S, Azar Y, Matzner Y (In press). The effect of serum amyloid A on selected in vitro functions of isolated human neutrophils. J Lab Clin Med
Hoffman JS, Benditt EP (1982) Changes of high density lipoprotein content following endotoxin administration in the mouse: formation of serum amyloid protein-rich subfractions. J Biol Chem 257:10510-10517
Kisilevsky R, Subrahmanyan L (1992) Serum amyloid A changes high density lipoprotein's cellular affinity. A clue to serum amyloid A's principal function. Lab Invest 66:778-785[Medline]
KluveBeckerman B, Drumm ML, Benson MD (1991) Nonexpression of the human serum amyloid A three (SAA3) gene. DNA Cell Biol 10:651-661[Medline]
KluveBeckerman B, Dwulet FE, Benson MD (1988) Human serum amyloid A. Three hepatic mRNAs and the corresponding proteins in one person. J Clin Invest 82:1670-1675[Medline]
KluveBeckerman B, Long GL, Benson MD (1986) DNA sequence evidence for polymorphic forms of human serum amyloid A (SAA). Biochem Genet 24:795-803[Medline]
Kouklis PD, Hutton E, Fuchs E (1994) Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins. J Cell Biol 127:1049-1060[Abstract]
Kushner I (1982) The phenomenon of the acute phase response. Ann NY Acad Sci 389:39-48[Medline]
Linke RP (1984) Monoclonal antibodies against amyloid fibril protein AA. Production, specificity, and use for immunohistochemical localization and classification of AA-type amyloidosis. J Histochem Cytochem 32:322-328[Abstract]
Linke RP, Bock V, Valet G, Rothe G (1991) Inhibition of the oxidative burst response of N formyl peptide-stimulated neutrophils by serum amyloid A protein. Biochem Biophys Res Commun 176:1100-1105[Medline]
Meek RL, Benditt EP (1986) Amyloid A gene family expression in different mouse tissues. J Exp Med 164:2006-2017[Abstract]
Meek RL, Benditt EP (1989a) Rat tissues express serum amyloid A protein-related mRNAs. Proc Natl Acad Sci USA 86:1890-1894[Abstract]
Meek RL, Eriksen N, Benditt EP (1989b) Serum amyloid A in the mouse. Sites of uptake and mRNA expression. Am J Pathol 135:411-419[Abstract]
Meek RL, UrieliShoval S, Benditt EP (1994) Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci USA 91:3186-3190[Abstract]
Poyart C, Wajcman H, Kister J (1992) Molecular adaptation of hemoglobin function in mammals. Respir Physiol 90:3-17[Medline]
PreciadoPatt L, Levartowsky D, Pras M, Hershkoviz R, Lider O, Fridkin M (1994) Inhibition of cell adhesion to glycoproteins of the extracellular matrix by peptides corresponding to serum amyloid A. Toward understanding the physiological role of an enigmatic protein. Eur J Biochem 223:35-42[Abstract]
Ramadori G, Sipe JD, Colten HR (1985) Expression and regulation of the murine serum amyloid A (SAA) gene in extrahepatic sites. J Immunol 135:3645-3647
Raynes JG, Eagling S, McAdam KP (1991) Acute-phase protein synthesis in human hepatoma cells. Differential regulation of serum amyloid A (SAA) and haptoglobin by interleukin-1 and interleukin-6. Clin Exp Immunol 83:488-491[Medline]
Sipe JD (1992) Amyloidosis. Annu Rev Biochem 61:947-975[Medline]
Sipe JD, Colten HR, Goldberger G, Edge MD, Tack BF, Cohen AS, Whitehead AS (1985) Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA. Biochemistry 24:2931-2936[Medline]
Steel DM, Donoghue FC, O'Neill RM, Uhlar CM, Whitehead AS (1996) Expression and regulation of constitutive and acute phase serum amyloid A mRNAs in hepatic and non-hepatic cell lines. Scan J Immunol 44:493-500[Medline]
Steel DM, Sellar GC, Uhlar CM, Simon S, DeBeer FC, Whitehead AS (1993) A constitutively expressed serum amyloid A protein gene (SAA4) is closely linked to, and shares structural similarities with, an acute-phase serum amyloid A protein gene (SAA2). Genomics 16:447-454[Medline]
Uhlar CM, Burgess CJ, Sharp PM, Whitehead AS (1994) Evolution of the serum amyloid A (SAA) protein superfamily. Genomics 19:228-235[Medline]
UrieliShoval S, Meek RL, Hanson RH, Eriksen N, Benditt EP (1994) Human serum amyloid A genes are expressed in monocyte/macrophage cell lines. Am J Pathol 145:650-660[Abstract]
UrieliShoval S, Meek RL, Hanson RH, Ferguson M, Gordon D, Benditt EP (1992) Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation. J Histochem Cytochem 40:1879-1885
Webb CF, Tucker PW, Dowton SB (1989) Expression and sequence analysis of serum amyloid A in the Syrian hamster. Biochemistry 28:4785-4790[Medline]
Whitehead AS, deBeer MC, Steel DM, Rits M, Lelias JM, Lane WS, deBeer FC (1992) Identification of novel members of the serum amyloid A protein superfamily as constitutive apolipoproteins of high density lipoprotein. J Biol Chem 267:3862-3867
Xu L, Badolato R, Murphy WJ, Longo DL, Anver M, Hale S, Oppenheim JJ, Wang JM (1995) A novel biologic function of serum amyloid A. Induction of T lymphocyte migration and adhesion. J Immunol 155:1184-1190[Abstract]
Yamada T, Kakihara T, Kamishima T, Fukuda T, Kawai T (1996) Both acute phase and constitutive serum amyloid A are present in atherosclerotic lesions. Pathol Int 46:797-800[Medline]
Zimlichman S, Danon A, Nathan I, Mozes G, ShainkinKestenbaum R (1990) Serum amyloid A, an acute phase protein, inhibits platelet activation. J Lab Clin Med 116:180-186[Medline]