Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity.

Apoptosis via the mitochondrial pathway requires release of cytochrome c into the cytosol to initiate formation of an oligomeric apoptotic protease-activating factor-1 (APAF-1) apoptosome. The apoptosome recruits and activates caspase-9, which in turn activates caspase-3 and -7, which then kill the cell by proteolysis. Because inactivation of this pathway may promote oncogenesis, we examined 10 ovarian cancer cell lines for resistance to cytochrome c-dependent caspase activation using a cell-free system. Strikingly, we found that cytosolic extracts from all cell lines had diminished cytochrome c-dependent caspase activation compared with normal ovarian epithelium extracts. The resistant cell lines expressed APAF-1 and caspase-9, -3, and -7; however, each demonstrated diminished APAF-1 activity relative to the normal ovarian epithelium cell lines. A competitive APAF-1 inhibitor may account for the diminished APAF-1 activity because we did not detect dominant APAF-1 inhibitors, altered APAF-1 isoform expression, or APAF-1 deletion, degradation, or mutation. Lack of APAF-1 activity correlated in some but not all cell lines with resistance to apoptosis. These data suggest that regulation of APAF-1 activity may be important for apoptosis regulation in some ovarian cancers.

Granzyme B-mediated apoptosis proceeds predominantly through a Bcl-2-inhibitable mitochondrial pathway.

Cytotoxic T lymphocytes kill virus-infected and tumor cell targets through the concerted action of proteins contained in cytolytic granules, primarily granzyme B and perforin. Granzyme B, a serine proteinase with substrate specificity similar to the caspase family of apoptotic cysteine proteinases, is capable of cleaving and activating a number of death proteins in target cells. Despite the ability to engage the death pathway at multiple entry points, the preferred mechanism for rapid induction of apoptosis by granzyme B has yet to be clearly established. Here we use time lapse confocal microscopy to demonstrate that mitochondrial cytochrome c release is the primary mode of granzyme B-induced apoptosis and that Bcl-2 is a potent inhibitor of this pivotal event. Caspase activation is not required for cytochrome c release, an activity that correlates with cleavage and activation of Bid, which we have found to be cleaved more readily by granzyme B than either caspase-3 or caspase-8. Bcl-2 blocks the rapid destruction of targets by granzyme B by blocking mitochondrial involvement in the process.

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Bcl-xL does not inhibit the function of Apaf-1.

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.

Calpain functions in a caspase-independent manner to promote apoptosis-like events during platelet activation.

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-delta. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, 'apoptotic' substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis.

Release of cytochrome c is important in many forms of apoptosis. Recent studies of CD95 (Fas/APO-1)-induced apoptosis have implicated caspase-8 cleavage of Bid, a BH3 domain-containing proapoptotic member of the Bcl-2 family, in this release. We now demonstrate that both receptor-induced (CD95 and tumor necrosis factor) and chemical-induced apoptosis result in a similar time-dependent activation of caspases-3, -7, -8, and -9 in Jurkat T cells and human leukemic U937 cells. In receptor-mediated apoptosis, the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. FMK), inhibits apoptosis prior to commitment to cell death by inhibiting the upstream activator caspase-8, cleavage of Bid, release of mitochondrial cytochrome c, processing of effector caspases, loss of mitochondrial membrane potential, and externalization of phosphatidylserine. However, Z-VAD.FMK inhibits chemical-induced apoptosis at a stage after commitment to cell death by inhibiting the initiator caspase-9 and the resultant postmitochondrial activation of effector caspases. Cleavage of Bid but not release of cytochrome c is blocked by Z-VAD.FMK demonstrating that in chemical-induced apoptosis cytochrome c release is caspase-independent and is not mediated by activation of Bid. We propose that caspases form an integral part of the cell death-inducing mechanism in receptor-mediated apoptosis, whereas in chemical-induced apoptosis they act solely as executioners of apoptosis.

Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.

Exit of cytochrome c from mitochondria into the cytosol has been implicated as an important step in apoptosis. In the cytosol, cytochrome c binds to the CED-4 homologue, Apaf-1, thereby triggering Apaf-1-mediated activation of caspase-9. Caspase-9 is thought to propagate the death signal by triggering other caspase activation events, the details of which remain obscure. Here, we report that six additional caspases (caspases-2, -3, -6, -7, -8, and -10) are processed in cell-free extracts in response to cytochrome c, and that three others (caspases-1, -4, and -5) failed to be activated under the same conditions. In vitro association assays confirmed that caspase-9 selectively bound to Apaf-1, whereas caspases-1, -2, -3, -6, -7, -8, and -10 did not. Depletion of caspase-9 from cell extracts abrogated cytochrome c-inducible activation of caspases-2, -3, -6, -7, -8, and -10, suggesting that caspase-9 is required for all of these downstream caspase activation events. Immunodepletion of caspases-3, -6, and -7 from cell extracts enabled us to order the sequence of caspase activation events downstream of caspase-9 and reveal the presence of a branched caspase cascade. Caspase-3 is required for the activation of four other caspases (-2, -6, -8, and -10) in this pathway and also participates in a feedback amplification loop involving caspase-9.

Pro-caspase-3 is a major physiologic target of caspase-8.

The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s-1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.

Epidermal growth factor-binding protein activates soluble and receptor-bound single chain urokinase-type plasminogen activator.

Epidermal growth factor-binding protein (EGF-BP) is a serine proteinase that reversibly associates with epidermal growth factor (EGF). We analyzed the reaction of EGF-BP with urokinase type plasminogen activator (u-PA), a serine proteinase that promotes pericellular proteolysis and cellular migration. EGF-BP cleaved single chain u-PA (scu-PA) between Lys158 and Ile159, converting the zymogen into enzymatically active two-chain u-PA (tcu-PA), as shown by SDS-PAGE, N-terminal sequence analysis, and enzymatic assay. The kcat and Km of the activation reaction were (5.6 +/- 0.6) x 10(-2)s-1 and 2.0 +/- 0.3 microM, yielding a catalytic efficiency of 2.8 x 10(4) M-1.s-1. EGF-BP also activated scu-PA bound to receptors on U937 monocytes as demonstrated by the generation of amidase activity against a tcu-PA-specific fluorogenic substrate. By activating scu-PA, EGF-BP may initiate u-PA-dependent cell surface proteolysis and therefore enhance EGF activities that require cellular migration and/or tissue remodeling.

Proteolytically active streptococcal pyrogenic exotoxin B cleaves monocytic cell urokinase receptor and releases an active fragment of the receptor from the cell surface.

Urokinase plasminogen activator (u-PA) receptor (u-PAR) is a glycosyl-phosphatidylinositol-anchored membrane protein that promotes pericellular proteolysis and cellular migration. This investigation demonstrates that u-PAR is a substrate for the proteolytically active form of streptococcal pyrogenic exotoxin B (SPE B), a potent virulence factor secreted by Streptococcus pyogenes. Treatment of U937 monocyte-like cells with SPE B decreased specific 125I-labeled single-chain u-PA binding by up to 85%. Cysteine proteinase inhibitors neutralized SPE B without affecting the activity of phosphatidylinositol-specific phospholipase C. Due to decreased u-PA binding, SPE B-treated U937 cells expressed decreased activity against a u-PA-specific fluorogenic substrate and plasminogen. SPE B released single-chain u-PA that was noncovalently bound to U937 cells or cross-linked to cellular receptors with bis(sulfosuccinimidyl) suberate. The mass of the released u-PA-receptor complex was 100 kDa. Western blot analysis confirmed that the u-PA receptor that was cleaved by SPE B is u-PAR. After deglycosylation, the mass of SPE B-released u-PAR was 35 kDa, slightly smaller than the phosphatidylinositol-specific phospholipase C-derived form of this receptor. SPE B-released u-PAR retained the ability to bind u-PA, as determined by u-PA affinity chromatography. We conclude that SPE B may inhibit u-PA binding to monocytic cells by at least two mechanisms: (i) by decreasing the level of functional cell surface u-PAR and (ii) by releasing a soluble form of u-PAR that competes with the cellular receptor for ligand.

Neurotrophin binding to human alpha 2-macroglobulin under apparent equilibrium conditions.

alpha 2-Macroglobulin (alpha 2M) is a broad-spectrum proteinase inhibitor and a carrier of certain growth factors. The purpose of this investigation was to characterize the interaction of alpha 2M with nerve growth factor-beta (NGF-beta), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and ciliary neurotrophic factor (CNTF) under apparent equilibrium conditions. Binding in solution was assessed using the cross-linking agent bis(sulfosuccinimidyl) suberate (BS3). Noncovalent binding of NGF-beta, NT-3, NT-4, and BDNF to native alpha 2M and alpha 2M-methylamine (a conformationally modified form of alpha 2M that is recognized by the alpha 2M receptor) reached apparent equilibrium in less than 20 min at 37 degrees C. Apparent KD values for the binding of NT-4, NGF-beta, NT-3, and BDNF to alpha 2M-methylamine were 61, 110, 120, and 150 nM, respectively. Native alpha 2M bound all four neurotrophins with decreased affinity. Unlabeled NGF-beta competed with the radioiodinated neurotrophins for binding to immobilized alpha 2M-methylamine. The K1 for unlabeled NGF-beta was 120 nM, in good agreement with the apparent KD determined by the BS3 method. The number of NGF-beta binding sites per immobilized alpha 2M-methylamine was 1.0. CNTF bound minimally, if at all, to native alpha 2M and alpha 2M-methylamine as determined using a number of techniques. The extent of binding was insufficient for the determination of an affinity constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolytic activation of single-chain precursor macrophage-stimulating protein by nerve growth factor-gamma and epidermal growth factor-binding protein, members of the kallikrein family.

Promacrophage-stimulating protein (MSP) is an 80-kDa protein that acquires biological activity after cleavage at an Arg-Val bond to a disulfide-linked alpha beta heterodimer by serine proteases of the intrinsic coagulation cascade. These proteases, which include serum kallikrein, factor XIIa and factor XIa, are members of the trypsin family of serine proteases. We now report that two other members of the family, nerve growth factor-gamma (NGF-gamma) and epidermal growth factor-binding protein (EGF-BP), cleave and activate pro-MSP to the disulfide-linked alpha beta heterodimer. Cleavage of 1.5 nM pro-MSP by 1 nM NGF-gamma or EGF-BP at 37 degrees C was almost complete within 30 min. These concentrations of enzyme are about 2 orders of magnitude less than is required for cleavage by serum kallikrein or factor XIIa. Cleavage of pro-MSP to MSP was associated with a conformational change in the protein, because the cleaved product, but not pro-MSP, was detected by a sandwich enzyme-linked immunoassay. Cleavage caused the appearance of biological activity, as measured by chemotactic activity of MSP for resident peritoneal macrophages, by MSP-induced macrophage shape change, and by stimulation of macrophage ingestion of C3bi-coated erythrocytes. These findings suggest the possibility of cooperative interactions between NGF-gamma or EGF-BP and pro-MSP in inflammation and wound healing.

Classification of alpha 2-macroglobulin-cytokine interactions based on affinity of noncovalent association in solution under apparent equilibrium conditions.

alpha 2-Macroglobulin (alpha 2M) binds numerous cytokines; however, since binding affinities have not been determined, it is difficult to compare various alpha 2M-cytokine interactions or predict whether alpha 2M-cytokine complexes will form in the presence of other cytokine-binding macromolecules. In this investigation, we used a novel method to demonstrate that transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 2, nerve growth factor-beta (NGF-beta), platelet derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and basic fibroblast growth factor (bFGF) reversibly associate with alpha 2M-methylamine to form noncovalent complexes. Apparent equilibrium was achieved in less than 15 min. Noncovalent alpha 2M-cytokine complexes were converted into covalent complexes; however, this occurred slowly. Therefore, a rapid equilibrium assumption was applied and equilibrium dissociation constants were determined using a single binding site model. KD values for the binding of cytokines to alpha 2M-methylamine varied by 2 orders of magnitude. The rank order of affinity was TGF-beta 2 (13 +/- 2 nM) > TGF-beta 1, NGF-beta > PDGF-BB > or = bFGF > TNF-alpha. Native alpha 2M bound TGF-beta 1, TGF-beta 2, NGF-beta, PDGF-BB, and TNF-alpha. Interferon-gamma did not bind to native alpha 2M or alpha 2M-methylamine. Each cytokine bound native alpha 2M with lower affinity than alpha 2M-methylamine except for TGF-beta 2 which bound both forms with equal affinity. In non-equilibrium systems, alpha 2M-methylamine appeared to bind more TGF-beta 2 due to the more rapid dissociation of TGF-beta 2-native alpha 2M complex. The classification of alpha 2M-cytokine complexes according to binding affinity should predict which complexes are most likely to form in cell culture and under various conditions in vivo.

Nerve growth factor-gamma activates soluble and receptor-bound single chain urokinase-type plasminogen activator.

Nerve growth factor-gamma (NGF-gamma) is a serine proteinase which reversibly associates with the well characterized neurotrophin NGF-beta. In this study, we demonstrated that NGF-gamma cleaves recombinant single chain urokinase-type plasminogen activator (scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the two u-PA chains were 33 and 22 kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleavage sites or further digestion of tcu-PA by NGF-gamma, even when conversion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa band was Ile-Ile-Gly-Gly-Glu, indicating that NGF-gamma cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage of scu-PA by NGF-gamma resulted in scu-PA activation. The kcat and Km for this reaction, as determined in a continuous assay with the tcu-PA-specific substrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S-2444), were (4.1 +/- 0.6) x 10(-2) s-1 and 2.3 +/- 0.4 microM, respectively. The catalytic efficiency (kcat/Km) for scu-PA activation by NGF-gamma was 1.3 x 10(4) M-1 s-1, compared with 6.2 x 10(5) M-1 s-1 for the activation of scu-PA by plasmin. NGF-gamma-cleaved scu-PA which was bound to receptors on U937 monocytoid cells. The apparent masses of the resulting u-PA cleavage products were identical to those generated in solution as determined by SDS-PAGE. Cell-associated scu-PA was activated by NGF-gamma, as determined by the generation of activity against the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-arginine-7-amino-4-methyl coumarin. By activating scu-PA, NGF-gamma may initiate the u-PA-dependent cell-surface proteinase cascade and support NGF-beta activities which involve cellular migration and/or extracellular matrix remodeling.

Regulation of macrophage alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma.

alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a broad specificity receptor that may function in lipoprotein metabolism, proteinase regulation, and growth factor regulation. In this study, we demonstrated that alpha 2M-R/LRP expression in macrophages can be markedly decreased by LPS and by IFN-gamma. Regulation of alpha 2M-R/LRP in RAW 264.7 cells was demonstrated at the mRNA, antigen, and receptor-function levels. In receptor-function studies, the decrease in alpha 2M-R/LRP expression was detected as a 90% decrease in the Bmax or maximum receptor binding capacity for activated alpha 2M after treatment with LPS or IFN-gamma. Western blot analysis of whole cell lysates demonstrated significant loss of alpha 2M-R/LRP heavy-chain. Northern blot analysis of poly(A)+ RNA revealed a marked decrease in alpha 2M-R/LRP mRNA after treatment with LPS (79% decrease) or IFN-gamma (70% decrease). Other cytokines, including tumor necrosis factor-alpha, transforming growth factor-beta-1, and interleukin-6 did not regulate alpha 2M-R/LRP. The ability of LPS and IFN-gamma to regulate alpha 2M-R/LRP was confirmed in experiments with primary cultures of murine bone marrow macrophages. These studies demonstrate that macrophage alpha 2M-R/LRP is subject to significant downregulation by physiologically significant cytokines and signaling macromolecules.

Reaction of nerve growth factor gamma and 7S nerve growth factor complex with human and murine alpha 2-macroglobulin.

The kallikrein-like serine proteinase nerve growth factor gamma (NGF-gamma) reacted with the plasma proteinase inhibitor human alpha 2-macroglobulin (h alpha 2M). The h alpha 2M subunits were cleaved, the electrophoretic mobility of h alpha 2M in nondenaturing polyacrylamide gels was increased, and the intrinsic fluorescence of h alpha 2M was increased with a slight blue-shift. These changes are well-characterized components of the alpha 2M/proteinase reaction mechanism. In N alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) hydrolysis experiments, the catalytic efficiency (kcat/KM) of the h alpha 2M-NGF-gamma complex was decreased by 98.5% compared with free NGF-gamma. This decrease is unique since other alpha 2M-proteinase complexes retain significant amidase activity. For comparison, we determined that the catalytic efficiency of alpha 2M-trypsin is decreased by 58% compared with free trypsin under equivalent conditions. The rate of NGF-gamma inhibition by h alpha 2M was (1.0 +/- 0.1) x 10(4) M-1 s-1 as determined by BAPNA hydrolysis. A similar value was determined by monitoring the change in intrinsic fluorescence. NGF-gamma, which was bound within the intact 7S NGF complex, also reacted with h alpha 2M, albeit at a very slow rate. This reaction may have depended exclusively on slow reversible dissociation of NGF-gamma from the 7S complex. NGF-gamma was rapidly inhibited by murine alpha 2M (m alpha 2M).(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and immunohistochemical localization of alpha 2-macroglobulin receptor (low-density lipoprotein receptor-related protein) in human brain.

Proteinase inhibitors have been implicated in brain development and in degenerative processes such as Alzheimer's disease. Low-density lipoprotein receptor-related protein (LRP) is a multifunctional cell-surface receptor that binds activated forms of the proteinase inhibitor, alpha 2-macroglobulin (alpha 2M) and apolipoprotein E. Solubilized plasma membranes of human cerebral cortical gray matter were subjected to affinity chromatography on alpha 2M-methylamine-sepharose. A single receptor was purified; this protein was LRP as determined by molecular mass, peptide structure, and immunoreactivity with monoclonal and polyclonal antibodies. In adult human brain, LRP immunoreactivity was abundant on neuronal cell bodies and proximal processes. Other cells within the neuropil, including glia and microvascular cells (endothelium and pericytes), were immunonegative. Weak LRP immunoreactivity was identified in a perivascular pattern corresponding to the location of astrocytic foot processes. The distribution of LRP in the central nervous system is consistent with the potential function of this receptor in the regulation of proteinase activity, cytokine activity, and cholesterol metabolism.