Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation.

Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

Ubiquitin profile in inflammatory leukocytes and binding of ubiquitin to murine AA amyloid: immunocytochemical and immunogold electron microscopic studies.

Lysosomes in activated murine monocytoid cells have been implicated in AA amyloid formation. The pathophysiology of this process is not well understood. Previous studies into the nature of the relationship between ubiquitin (UB), possessing intrinsic amyloid enhancing factor (AEF) activity; serum amyloid A (SAA), the precursor protein of AA amyloid; and activated monocytoid cells have indicated a temporal and spatial relationship between these proteins and tissue AA amyloid deposits. To extend these findings, we have examined murine peritoneal leukocytes and splenic tissues during the early amyloid deposition phase by immunocytochemical and immunogold electron microscopic methods using monospecific anti-ubiquitin and anti-mouse AA amyloid antibodies. We show here enrichment of endosome-lysosome-like (EL) vesicles in the activated monocytoid cells with UB and SAA, and the presence of UB-bound AA amyloid fibrils in the EL vesicles, perikarya, and interstitial spaces. The importance of these findings is emphasized by the fact that activated monocytoid cells, containing UB in the EL vesicles, sequester and eventually localize SAA in their EL vesicles, and that UB binds to the EL-contained AA amyloid fibrils. These findings may also have functional consequences for studies on the role of EL and UB in amyloidogenesis.

Ubiquitin: its potential significance in murine AA amyloidogenesis.

Amyloid enhancing factor (AEF), which has recently been shown to have identity with ubiquitin (Ub), is believed to play a causative role in experimentally induced AA amyloidosis in mice. We have examined the profile of Ub in activated leukocytes and splenic reticulo-endothelial (RE) cells and its relationship with serum amyloid A protein (SAA) and AA amyloid deposits in an alveolar hydatid cyst (AHC)-infected mouse model of AA amyloidosis. Two monospecific antibodies, anti-ubiquitin (RABU) and anti-mouse AA amyloid, were used as immunological probes to localize Ub, SAA, and AA amyloid. In response to AHC infection, the dull and diffuse Ub immunoreactivity in normal mouse leukocytes and RE cells promptly changed to a discrete granular pattern suggesting an increase in the intracellular concentration of Ub and the formation of Ub-protein conjugates. This corresponded to an elevation in SAA levels, SAA uptake by RABU-positive phagocytic cells, co-localization of Ub-SAA immunoreactive splenocytes in the perifollicular areas, and deposition of Ub-bound AA amyloid in the splenic and hepatic tissues. These results suggest that Ub-loaded monocytoid cells may play an important role in the physiological processing of the sequestered SAA into AA amyloid. Aspects of AA amyloidogenesis are discussed in relation to other experimental models in which stress-induced Ub-protein conjugate formation and its transport to lysosomal vesicles have been studied.

Ubiquitin profile and amyloid enhancing factor activity in Alzheimer and 'normal' human brain extracts.

Tris-HCl or Laemmli sample buffer extracted frontal lobe and hippocampal samples from normal aged and Alzheimer's disease (AD) subjects were used to determine total ubiquitin (Ub), distribution of monomeric Ub and Ub-protein conjugates and amyloid enhancing factor (AEF) activity using the dot-blot, Western blot and mouse AEF bioassay techniques, respectively. The AD samples, as compared to the normals, demonstrated a 1.7-fold increase in total Ub, elevated levels of Ub-protein conjugates and an appreciably enhanced AEF activity. Many of the hippocampal Ub-protein conjugates were found to be soluble only in the Laemmli sample buffer. The possible roles of elevated Ub levels and of the association of AEF activity with Ub are discussed in regard to pathogenesis of brain amyloidosis.

Amyloid enhancing factor activity is associated with ubiquitin.

Crude amyloid enhancing factor (AEF) drastically reduces the pre-amyloid phase on passive transfer and induces amyloid deposition in the recipient mice in 48-120 h. We attempted to purify AEF from murine amyloidotic liver and spleen extracts by using gel filtration, preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis and ion exchange chromatography and isolated a 5.5 kDa peptide. In the mouse bioassay, this peptide induced accelerated splenic AA deposition in a dose-dependent manner. Based on structural, electrophoretic and immunochemical criteria the peptide was identified as ubiquitin. A polyclonal rabbit anti-bovine ubiquitin IgG antibody (RABU) abolished the in vivo AEF activity of crude murine AEF in a dose-dependent manner. Monomeric ubiquitin and its large molecular weight adducts were isolated from crude AEF using cyanogen bromide-activated sepharose conjugated to RABU and size exclusion chromatography methods. These were assayed and were found to possess AEF activity. Furthermore, increased levels of ubiquitin, a phenomenon similar to that of AEF, were detected by immunocytochemistry in mouse peritoneal leucocytes prior to and during amyloid deposition. Since AEF shares a number of biological and functional properties with ubiquitin, we suggest a possible role of ubiquitin as an AEF, and that serum amyloid protein A and ubiquitin, the two reactants generated during inflammatory stress conditions, may converge to induce AA amyloid deposition.

Binding of ubiquitin to experimentally induced murine AA amyloid.

Amyloid enhancing factor (AEF) activity has recently been demonstrated in ubiquitin purified from amyloidotic murine tissues and Alzheimer brain extract. Since AEF is known to bind to amyloid fibrils and 'fibril-AEF' on passive transfer induces accelerated amyloidogenesis in the recipient animals, it was of interest to investigate whether ubiquitin binds to amyloid. Immunohistological studies were carried out on liver sections from amyloidotic mice. Biotin-strepavidin-peroxidase methods using monospecific rabbit anti-mouse AA amyloid IgG (RAAG) and rabbit anti-bovine ubiquitin IgG (RABU) antibodies were employed to immunostain the amyloid and ubiquitin deposits, respectively. RABU-treated liver sections were counterstained with thioflavine S. RAAG reacted strongly with the amyloid, indicating that it is AA type, and RABU-positive immunodeposits were found bound to the thioflavine-S-positive AA deposits. Treatment of the liver sections with 0.1 M sodium acetate containing 0.5 M NaCl, pH 4, for 2-3 h at 37 degrees C nearly completely desorbed the AA amyloid-bound ubiquitin. Since ubiquitin demonstrates AEF activity in vivo and binds non-covalently to AA amyloid, we suggest that ubiquitin may indeed be 'fibril-AEF' and may play a crucial role in the pathogenesis of amyloidosis. To our knowledge, this is the first time that ubiquitin bound to extracellularly deposited amyloid has been demonstrated.

Alzheimer's disease brain-derived ubiquitin has amyloid-enhancing factor activity: behavior of ubiquitin during accelerated amyloidogenesis.

Amyloid-enhancing factor (AEF) is believed to be a crucial common pathogenetic link in diverse forms of human amyloidosis. Passive transfer of crude AEF is known to trigger accelerated splenic amyloid deposition in mice. We have recently identified AEF activity in ubiquitin isolated from murine amyloidotic tissues. Using similar techniques we have purified ubiquitin, from crude Alzheimer's disease (AD) brain extracts, to apparent homogeneity. Based on the partial amino acid sequence homology, immunochemical and pathophysiological criteria, the approximately 5.5-kDa AD-derived protein was identified as ubiquitin (AD-ubiquitin) with AEF activity. Ten to twenty micrograms of this protein per mouse, with or without CaCl2, in conjunction with four subcutaneous injections of 0.5 ml of 1% aqueous AgNO3, induced accelerated splenic amyloid deposition. By immunohistochemistry, using anti-mouse AA amyloid antibody, the AD-ubiquitin-induced amyloid was identified as AA type. With anti-bovine ubiquitin antibody, using similar spleen sections as above, ubiquitin was found to co-deposit with AA amyloid in the splenic perifollicular areas. These results strongly suggest that ubiquitin may be involved in the pathogenesis of amyloidosis.

Biochemical nature and cellular origin of amyloid enhancing factor (AEF) as determined by anti-AEF antibody.

Low ionic strength acidic buffer, Sephadex G-200 and Benzamidine-Sepharose (BZ) gel chromatography, have been used for the partial purification of alveolar hydatid cyst (AHC) induced amyloid enhancing factor (AEF). BZ-gel bound AEF (AEF-BZ) demonstrated AEF activity in the mouse bioassay, proteolytic activity against Hide powder azure showed two major and three minor peptides on SDS-PAGE. Pretreatment of AEF-BZ with 10 mM phenylmethylsulphonyl fluoride or 20 mM p-chloromercuribenzoic acid completely abolished its bioactivity in vivo and proteolytic activity in vitro. Polyclonal anti-AEF antibody (AAA) was generated which on passive transfer into mice completely abolished the bioactivity of both casein-induced, or AHC-induced AEF. The AAA absorbed on Sepharose gel conjugated to normal mouse serum developed one common precipitin band between AE and AEF-positive sera from AHC-infected and old retired mice and in immunostaining it bound to the cytoplasmic granular components of a majority of splenic and peritoneal leucocytes from AHC-infected mice. In contrast, only a few normal mouse leucocytes showed positive staining. We suggest that AEF, in all probability, is a serine/thiol protease of leucocyte origin whose intracellular and humoral concentrations increase significantly during amyloidosis. The role of lysosomal proteases and anti-AEF antibody which has been successfully generated for the first time is discussed with reference to the origin of AEF and its presumed biological function in amyloidogenesis.

Evidence for increased amyloid enhancing factor activity in Alzheimer brain extract.

Soluble brain extracts containing 0.1 to 16 mg of protein from 3 normal human brain and 11 patients with Alzheimer's disease, Down's syndrome and other neurological disorders were assayed for amyloid enhancing factor (AEF) activity in the mouse bioassay. At the 0.1 mg dosage, five of seven brain extracts from amyloid-positive samples and only one of four amyloid-negative samples demonstrated AEF activity. Marginal AEF activity was detected in the normal brain extracts at 8 or 16 mg protein dosage. Alzheimer-AEF was aggregated by exhaustive dialysis against 0.01 M phosphate buffer, pH 6 or distilled water and the solubilized aggregate was fractionated on a BioGel P-60 column. Of the two protein peaks, AEF activity was present only in the low mol.wt second fraction, which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed two discrete and three minor peptide bands between 60 and 66 kDa and one of these was periodic acid-Schiff positive, and three fuzzy bands near 14 kDa. Pretreatment of the crude and second fraction with 10 mM phenylmethylsulfonyl fluoride (PMSF) nearly completely abolished the in vivo AEF bioactivity. It is suggested that (a) a higher AEF concentration is present in amyloid-positive brain samples than those negative for amyloid or normal brain tissues, (b) AEF-positive fraction contains at least five dominant peptides ranging between 14 to 66 kDa, and (c) abolition of PMSF-treated Alzheimer-AEF activity, similar to that of murine AEF, might be due to its serine/thiol proteinase nature. To our knowledge, this is the first time that AEF activity has been demonstrated in Alzheimer brain samples.