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Serum amyloid P and related mble&ks-associ-ated with the acute-phase response in Alzheimer’s disease R.N. Departments

of Neurology

and Neurosciences, Cleveland,

Kalaria Case Western Reserve University OH 44106 (USA)

Serum amyloid P (SAP) is a unique glycoprotein that has long been of interest as an acute-phase protein. SAP is normally present in plasma in a discoid pentagonal conformation made up of 10 noncovalently bound subunits, that characterizes it as a pentraxin (Pepys et al., 1982; Osmand et al., 1977; Skinner and Cohen, 1988). The pentameric structure is also shared by another glycoprotein, the C-reactive protein (CRP). The plasma concentrations of SAP and CRP are differentially regulated during the course of inflammation in a wide variety of animals (Pepys et al., 1982). Although SAP is clearly identified with the acute-phase response in mice (Le et al., 1982; Murakami et al., 1988; Pepys ef al., 1982), its exact function in the human body still remains elusive. There are some indications that it may be involved in a late response related to chronic illness (Murakami et al., 1988; Pepys, 1986). On the other hand, CRP is a major acute-phase protein which increases markedly within hours of onset of tissue injury or necrosis in man (Kushner, 1982). Human SAP and CRP not only share the pentameric structure but also extensive amino acid sequences and their genes are closely located on the same arm of chromosome 1 (Mantzouranis et al., 1985 ; Lei et al., 1985). There are differences, however, in their chemical and immunological properties with respect to ligand-binding specificities and glycosylation patterns (Pepys et al., 1982). The specific binding properties of SAP and its conservation in various species (Vasta, 1990) suggest that it has important functions. SAP is now known to be associated with almost all forms of amyloidosis (Glenner, 1980) and it is this feature that makes it an interesting molecule (Kalaria et al., 1991a) to investigate in Alzheimer’s disease (AD). Serum amyloid

P component

in amyloidosis

In 1965, Cohen and colleagues (Cathart et al., 1965) reported the extraction of an u-globulin termed

Correspondence Cleveland, Ohio

to: 44106

Dr. Rajesh (USA).

N.

Kalaria,

Department

of

School of Medicine,

amyloid P (AP) component (P for plasma) associated with amyloid fibrils present in peripheral deposits. Subsequent studies confirmed that SAP was the same as a normal cc,-glycoprotein (Glennei, 1980) or complement subcomponent Cl t described by Osmand et al. (1977) and that AP extracted from amyloid deposits is indistinguishable from circulating SAP in terms of antigenicity, molecular weight, appearance and the primary structure, as well as binding properties to amyloid fibrils (Baltz et al., 1986; Skinner and Cohen, 1988). Circulating SAP continuously deposits from blood into peripheral amyloid deposits, and the bound molecule is presumably in dynamic equilibrium with the circulating SAP (Hawkins et al., 1988a). Pepys and colleagues (Hawkins et al., 1988b, 1990) have used this clinically to initiate the concept of targeting and imaging of peripheral amyloid deposits with iodinated AP. Indeed, if this technique can be resolved to locate cerebral amyloid deposits, then interesting possibilities may open up. Until recently, SAP was considered to be absent from the intracerebral amyloid deposits of AD and other neurodegenerative diseases (Pepys, 1988). Previous attempts to demonstrate SAP in intracerebral amyloid deposits of AD subjects were unsuccessful (Rowe et al., 1984; Westermark et al., 1982). In these earlier studies, positive AP antigenicity was evident only in cerebral vessels (Rowe et al., 1984), where SAP presumably binds to disrupted vessels. More recently, however, SAP immunoreactivity was described in cortical amyloid plaques of AD (Coria et al., 1988; Yamada et al., 1987) and other neurodegenerative disorders (Castano and Frangione, 1988; Kalaria et al., 1991a). In addition, we (Kalaria and Grahovac, 1990; Kalaria et al., 1991a) and others (Duong et al., 1989) confirmed that SAP is evident in the neurofibrillary pathology of AD and also in thioflavin-S-positive tangles of the choroidal epithelial cells (Kalaria, unpublished observations). This finding indirectly gives support to there being anti-

Neurology,

University

Hospitals

of Cleveland,

2074

Abington

Road,

45th FORUM

638

IN IA4MUNOLOGY

unravel any spatial relationships between SAP immunoreactivity and sites of amyloid deposition, we first considered the origins of SAP in brain amyloid deposits. It was previously reported that SAP is synthesized by hepatocytes, liver being the only tissue that expresses its mRNA in normal human subjects (Ohnishi et al., 1986). This is in keeping with the significant reduction of the molecule in hepatic disease (Pepys et al., 1978). To confirm the liver origin of SAP and consider the possibility that SAP might be synthesized in situ by reactive parenchymal cells such as astrocytes, we searched for its mRNA employing the more sensitive polymerase chain reaction technique (Kalaria et al., 199lb,c). Using 3 different pairs of primers and RNA isolated from pooled brain tissue from a large number of subjects, we found no evidence that SAP mRNA is expressed by brain (table I) or any other organ tested except the liver (Kalaria et al., 1991~). These findings strongly imply that this relatively large protein and possibly other yet uncharacterized proteins involved in immune mechanisms originate from the circulation perhaps through a leaky endothelium exacerbated by the release of cytokines (Bauer et al., 1991; Eikelenboom et al., 1991) from perivascular macrophages during the disease process. It is conceivable that such increased permeability or porosity of the cerebral endothelium could occur locally and transiently over a protracted period of time. The broader implications of these findings, of course, relate to the integrity of the blood-brain barrier (BBB) in AD. This issue is of much interest though controversial and pertains to the vascular origin of cerebral P-amyloid deposition (Castano and Frangione, 1988; Pepys, 1988). The localization of SAP in cerebral amyloid presumably occurs through a slow process different from that involved in the relatively rapid localization

genie similarities and a link between amyloid deposits of cerebral vessels, senile plaques and neurofibrillary tangles. Nevertheless, the most important conclusion these recent studies have established is that SAP is also present in cerebral amyloid deposits including diffuse plaques (table I), which do not apparently contain serum amyloid A, transthyretin or P,-microglobulin, proteins invariably regulated during the acute phase and involved in other types of amyloids (Glenner, 1980; Kalaria et al., 1991a; Pepys, 1988).

Significance of SAP in AD

The role of SAP in cerebral amyloidosis as related to AD is unknown. Neither the mechanism involved in the localization or binding of SAP to cerebral amyloid deposits nor the temporal relationship between SAP accumulation and amyloid deposition is clear. Although it is possible that the presence of SAP in cerebral amyloid deposits is an epiphenomenon, with no functional consequences, Pepys (1988) has suggested an interesting alternative. The presence of AP may contribute in some way to the deposition and persistence of amyloid deposits. SAP may serve a “protective” role in amyloid deposition, shielding the fibrils from degradation by acting as a protease inhibitor (e.g. elastase or an as yet unknown lysosomal enzyme) or by preventing them from stimulating or activating phagocytic cells. However, akin to the intensive study of the ADlinked P-amyloid precursor protein of which the normal function is also unknown, understanding the role of SAP in AD may be important. In an attempt to elucidate these mechanisms and

Table I. Expression of acute-phase proteins in Alzheimer

Detection

brain.

in brain lesions

Acute-phase protein

SP

NFT

Vessels

mRNA

Amyloid P component C-reactive protein a,-Antichymotrypsin a,-Antitrypsin Antithrombin-III u,-Macroglobulin Inter a,-trypsin inhibitor Complement proteins (Clq, C3, C4) Complement-C4-binding protein

+ f + + + -I+ + +

+ 0 + + + & f 0 -

•k 0 Ik + It 0 0 f +

0 ND

Results taken from Bauer et a/., 1991; Eikelenboom Kalaria, unpublished results; McGeer ef a/., 1990. SP = senile plaques; NFT = neurofibrillary tangles;

er a/.,

1991 ; Gollin

+ = presence;

et al., 0 = absence;

1992;

Kalaria + = weak

and

Kroon,

staining;

ND + ND ND

1991 ; Kalaria ND

= not

el al.,

determined.

199lb.c;

IMMUNOLOGICAL

FACTORS

of AP in systemic amyloids (Hawkins et al., 1988b). In addition to simple leakage, the possibility that the protein is selectively transported by mechanisms within the cerebral endothelium or the choroidal epithelium, or conveyed by extracellular receptors on blood-derived macrophages or immune responsive cells (Siripont et al., 1988) that translocate within brain, also needs to be evaluated. While these possibilities have not been fully examined, the specific binding of SAP to polyanions may implicate such mechanisms (Pepys et al., 1982). The binding properties of SAP have direct relevance to the immunological mechanisms in amyloid deposition. Apart from amyloid fibrils, SAP binds to other molecules common to basal lamina, including fibronectin and some polyanions (Pepys et al., 1982). It has been established that SAP exhibits calcium-dependent binding to heparan and dermatan sulphates (Hamazaki, 1987) which have been found to be common constituents of amyloid deposits (Kisilevsky and Snow, 1988; Perry et al., 1991). Further clues to realizing the significance of SAP in cerebral amyloid deposits may be provided by considering similarities between peripheral amyloidotic lesions and those in brain (Kisilevsky and Snow, 1988). Interestingly, the presence of inorganic complexes such as aluminosilicates or calcium phosphate (Kula et al., 1977) is another feature that is invariably common to amyloid deposits. Thus the localization of SAP is concomitant with the immobilization of calcium and the association of highly sulphated GAG with the amyloid fibrils (Perry et al., 1991). Recent studies indicate that SAP displays specific binding to complement proteins in particular complement-C4-binding protein (Schwalbe et al., 1990) which is involved in the inhibition of the classical complement cascade (table I). Consistent with this, we have observed that SAP is co-localized with the complement-C4-binding protein in neocortical amyloid plaques (unpublished observations). The latter observation suggests there is close interaction between the complement proteins and SAP during the pathogenesis. This is also supported by the observation that both SAP and complement protein C3 are found in diffuse plaques in the absence of other molecules (Kalaria et al., 199la). The specific binding and interaction of SAP (and possibly other molecules) with amyloid may also explain why the more common serum proteins do not show the same distribution and localization patterns (Alafuzoff et al., 1987; Rozemuller et al., 1988) irrespective of whether these originate from the circulation across a breached BBB (Wisniewski and Kozlowski, 1982). However, the BBB also seems. to be affected in non-demented aging controls (Alafuzoff et al., 1987), which may merely reflect that the degree of BBB impairment is related to the severity of the pathology, i.e. in agingperse, the BBB

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639

is less altered. Also, proteins that gain entry into brain may be differentially sequestered (Mann et al., 1986) and metabolized by microglia or macrophages, while those which specifically interact with amyloid or amyloid formation, like SAP, may be resistent to proteolysis (Pepys, 1986). This notion would be in agreement with the fact that the specific localization and binding of SAP to Alzheimer lesions is concomitant with the pathogenetic process rather than a result of agonal state or postmortem factors (Kalaria et al., 1991a; Mori et al., 1991; Pouplard-Barthelaix, 1988). Taken together, these observations do not necessarily negate that the BBB is breached in AD. Most surprisingly, however, these approaches have led to the discovery that mRNA of many classical acute-phase serum proteins including the complement proteins (Rozemuller et al., 1988; McGeer et al., 1990), ul-antichymotrypsin (Pasternack et al., 1990) and antithrombin III (Kalaria and Kroon, 1991) localized in amyloid deposits (Kalaria et al., 199la; McGeer et al., 1990) are readily expressed in both normal and AD brain tissue. The localization of mRNA of these acute-phase proteins (table I) in brain tissue strongly implicates that the brain has the remarkable capacity to sythesize and execute an immune response. This is the most enlightening outcome of these studies that may indicate another fundamental characteristic of the brain during injury and its potential plasticity. C-reactive protein Although SAP and CRP are considered members of the same plasma protein family, CRP is not recognized to be associated with amyloid deposits (Baltz et al., 1982). This is consistent with the different behaviours of the two pentraxins in humans. Whilst most studies to date also suggest it to be absent from brain amyloid deposits (Duong et al., 1989; Kalaria et al., 199la; Rozemuller et al., 1989), upon reexamination of some AD cases with severe pathology, we have recently observed weak CRP-like immunoreactivity in cortical amyloid deposits (table I). It remains to be tested whether CRP is only produced in the liver though our preliminary findings may have similar implications as SAP in AD, discussed above. Acute-phase proteins,

the serpins and SAP

Cerebral amyloid deposits have also been found to contain other acute phase proteins (table I) such as a,-macroglobulin (Bauer et al., 1991), the serpins and complement proteins (Eikelenboom et al., 1991). We recently detected a,-antitrypsin and antithrombin-111 in the cerebral amyloid deposits and neurofibrillary pathology of AD (Gollin et al., 1992; Kalaria and Kroon, 1991). However, there is no

640

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general consensus that other less known acute-phase proteins such as the immunoglobulins, albumin and transferrin are localized in amyloid deposits although there is evidence that most of these proteins may be produced intrathecally (Kalaria et al., 1991c). It is noteworthy, however, that a,-antitrypsin, ceruloplasmin, the complement proteins C3 and C4 and the properdin factor B were significantly elevated in sera of AD subjects compared to age-matched healthy controls (Giometto et al., 1988). In this study, there were no changes in concentrations of other acutephase proteins including CRP. Nevertheless, these observations support the involvement of the immune system response, albeit locally, in the pathogenesis of AD. Thus, it is reasonable to suggestthat though SAP might not behave as a “true” acute-phase protein, it plays some role in the establishment of the inflammatory response,e.g. complement fixation or protease inhibitor function, and therefore in the pathogenesis of cerebral amyloidosis.

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