Selective liver targeting of antivirals by recombinant chylomicrons--a new therapeutic approach to hepatitis B.

Hepatitis B virus (HBV) infection is the world's most important chronic virus infection. No safe and effective treatment is available at present, and clinical exploration of promising antiviral agents, such as nucleoside analogues is hampered because of significant side-effects due to their aspecific body distribution. We are exploring the possibility of the selective delivery of antiviral active drugs to liver parenchymal cells, the main site of infection and replication of HBV. Chylomicrons, which transport dietary lipids into the liver via apolipoprotein E-specific receptors, could serve as drug carriers. However, their endogenous nature hampers their application as pharmaceutical drug carriers. We report here that incorporation of a derivative of the nucleoside analogue iododeoxyuridine into recombinant chylomicrons leads to selective targeting to liver parenchymal cells. Potentially effective intracellular drug concentrations of 700 nM can be achieved, and we therefore anticipate that these drug carrier complexes represent a conceptual advance in the development of an effective and safe therapy for hepatitis B.

Crosslinking of surface immunoglobulin and Fc receptors on B lymphocytes inhibits stimulation of inositol phospholipid breakdown via the antigen receptors.

F(ab')2 fragments of rabbit anti-mouse Ig induce proliferation of murine B lymphocytes, whereas the intact antibodies are not mitogenic. F(ab')2 anti-Ig stimulates the rapid breakdown of inositol phospholipids in B cells, resulting in the prolonged release of inositol (poly)phosphates and diacylglycerol. In marked contrast, intact anti-Ig initially induces a comparable response, which is abrogated after some 30 s. Blocking either the Fc receptors on the B cells or the Fc portion of the antibodies significantly reversed the inhibitory effect. On the other hand, both forms of anti-Ig elicited comparable increases in free cytoplasmic Ca2+ levels in B cells. These results therefore indicate that crosslinkage of Fc and surface Ig receptors on B cells inhibits inositol phospholipid breakdown (but not Ca2+ flux) resulting from ligation of the antigen receptors. Since there is evidence implicating inositol phospholipid breakdown in the induction of cell growth, this effect could provide a biochemical explanation for the known capacity of antigen-antibody complexes to inhibit B cell activation.

Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation.

Several studies have shown improved efficacy of cholesteryl-conjugated phosphorothioate antisense oligodeoxynucleotides. To gain insight into the mechanisms of the improved efficacy in vivo, we investigated the disposition of ISIS-9388, the 3'-cholesterol analog of the ICAM-1-specific phosphorothioate oligodeoxynucleotide ISIS-3082, in rats. Intravenously injected [(3)H]ISIS-9388 was cleared from the circulation with a half-life of 49.9 +/- 2.2 min (ISIS-3082, 23.3 +/- 3.8 min). At 3 h after injection, the liver contained 63.7 +/- 3. 3% of the dose. Compared to ISIS-3082, the hepatic uptake of ISIS-9388 is approximately 2-fold higher. Endothelial, Kupffer and parenchymal cells accounted for 45.7 +/- 5.7, 33.0 +/- 5.9 and 21.3 +/- 2.6% of the liver uptake of [(3)H]ISIS-9388, respectively, and intracellular concentrations of approximately 2, 75 and 50 microM, respectively, could be reached in these cells (1 mg/kg dose). Preinjection with polyinosinic acid or poly-adenylic acid reduced the hepatic uptake of [(3)H]ISIS-9388, which suggests the involvement of (multiple) scavenger receptors. Size exclusion chromatography of mixtures of the oligonucleotides and rat plasma indicated that ISIS-9388 binds to a larger extent to high molecular weight proteins than ISIS-3082. Analysis by agarose gel electrophoresis indicated that ISIS-9388 binds more tightly to plasma proteins than ISIS-3082. The different interaction of the oligonucleotides with plasma proteins possibly explains their different dispositions. We conclude that cholesterol conjugation results in high accumulation of phosphorothioate oligodeoxynucleotides in various liver cell types, which is likely to be beneficial for antisense therapy of liver-associated diseases.

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. II. Effect of charge.

Experiments presented in this paper suggest that sinusoidal rat liver cells recognize basic groups on proteins and that this recognition results in endocytosis of the proteins. Evidence for involvement of basic groups was obtained in two ways. Firstly, we changed the positively charged amino groups of the cross-linked ribonuclease molecules to neutral or negative by acetylation or succinylation, respectively. The modified proteins did not contain easily reducible disulfide bonds and they were not very sensitive to endoproteases, suggesting that they were not denatured by the acetylation procedures. Acetylation and succinylation reduced uptake of the injected cross-linked ribonuclease derivatives by liver and spleen and abolished their rapid clearance from plasma. In nephrectomized rats about 75% of the polymer, 36% of the acetylated polymer and 32% of the succinylated polymer were endocytosed by liver after 6 h. For the dimer fractions these values were 59%, 23% and 27%, respectively. Autoradiography and subcellular fractionation of liver 30 min post-injection localized the acetylated polymer in the lysosomal/microsomal fraction of sinusoidal liver cells, probably endothelial cells. Secondly, a positive correlation was found between binding of a number of ribonuclease derivatives to the cation exchanger SP-Sephadex G-25 and the rate of endocytosis by sinusoidal liver cells.

Recombinant lipoproteins: lipoprotein-like lipid particles for drug targeting.

Lipoproteins are endogenous particles that transport lipids through the blood to various cell types, where they are recognised and taken up via specific receptors. These particles are, therefore, excellent candidates for the targeted delivery of drugs to various tissues. For example, the remnant receptor and the asialoglycoprotein receptor (ASGPr), which are uniquely localised on hepatocytes, recognise chylomicrons and lactosylated high density lipopoteins (HDL), respectively. In addition, tumour cells of various origins overexpress the low density lipoprotein (LDL) receptor that recognises apolipoprotein E (apoE) on small triglyceride-rich particles and apoB-100 on LDL. Being endogenous, lipoproteins are biodegradable, do not trigger immune reactions, and are not recognised by the reticuloendothelial system (RES). However, their endogenous nature also hampers large-scale pharmaceutical application. In the past two decades, various research groups have successfully synthesised recombinant lipoproteins from commercially available natural and synthetic lipids and serum-derived or recombinant apolipoproteins, which closely mimic the metabolic behaviour of their native counterparts in animal models as well as humans. In this paper, we will summarise the studies that led to the development of these recombinant lipoproteins, and we will address the possibility of using these lipidic particles to selectively deliver a wide range of lipophilic, amphiphilic, and polyanionic compounds to hepatocytes and tumour cells. In addition, the intrinsic therapeutic activities of recombinant chylomicrons and HDL in sepsis and atherosclerosis will be discussed.

Water-soluble cholesteryl-containing phosphorothioate monogalactosides: synthesis, properties, and use in lowering blood cholesterol by directing plasma lipoproteins to the liver.

The synthesis of several monogalactoside-terminated phosphorothiolated cholesteryl derivatives is described. Monogalactosyl derivatives are coupled by phosphorothiolation to cholesterol by using ethylene glycol units as hydrophilic spacer moieties. The resulting compounds are easily soluble in water. Upon addition of such solutions to human serum (to 2 mM final concentration) the compounds are readily incorporated into lipoproteins. Isolated low-density lipoprotein (LDL) and high-density lipoprotein (HDL), preloaded with the compounds, are rapidly cleared from the circulation by the liver. The hepatic association is blocked by N-acetylgalactosamine, which indicates that galactose-specific recognition sites are responsible for the increased liver uptake. The plasma clearance and hepatic uptake of LDL loaded with the compounds is substantially higher (about 2-fold) than clearance and uptake of HDL containing the compounds. The selectivity of the effects of monogalactoside-terminated phosphorothiolated cholesteryl derivatives on the in vivo behavior of LDL as compared to that of HDL indicates that these compounds might be used to lower specifically LDL levels in patients with a high LDL-cholesterol level.

Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor.

A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells. In contrast, a glycolipid content of 50% (w/w) led to a liver uptake that could not be inhibited by preinjection with asialofetuin, indicating that the liposomes were now processed by the Gal/Fuc-recognizing receptor on liver macrophages. The results presented in this study are important for future targeting of water-soluble and amphiphilic drugs, enveloped in these glycolipid-laden liposomes, to parenchymal liver cells.

Specific targeting of a lipophilic prodrug of iododeoxyuridine to parenchymal liver cells using lactosylated reconstituted high density lipoprotein particles.

We recently reported the conversion of the water-soluble antiviral drug iododeoxyuridine (IDU) into the lipophilic prodrug dioleoyl-iododeoxyuridine (IDU-Ol2). The prodrug was incorporated into reconstituted high-density lipoprotein (NeoHDL) particles with physical and biological properties similar to those of native HDL. We also found, in initial experiments, that lactosylation of the prodrug-loaded NeoHDL increases its liver uptake. Because this offers the attractive perspective of using these particles for the delivery of drugs to the liver, we now analyze the characteristics and biological fate of lactosylated IDU-Ol2-loaded NeoHDL. The particles (containing approximately 25 prodrug molecules) have the same size and charge as native HDL, indicating that lactosylation does not cause aggregation or oxidative modification. At 10 min after intravenous injection of lactosylated [3H]IDU-Ol2-loaded NeòHDL into rats, only 13.5 +/- 2.8% of the dose was left in plasma and 75.9 +/- 2.4% of the dose was recovered in the liver. The relative specific uptake by the liver was 1-2 orders of magnitude higher than that of any other tissue. The hepatic uptake of lactosylated [3H]IDU-Ol2-loaded NeoHDL was much higher than that of free [3H]IDU ( < 20% of the dose). Both parenchymal liver cells and Kupffer cells express galactose-specific receptors. By isolating liver cells after injection of the prodrug-loaded particles, it was established that hepatic uptake occurred mainly (for 84.4 +/- 3.8%) in parenchymal liver cells. Preinjection with asialofetuin substantially reduced the liver uptake of lactosylated [3H]IDU-Ol2-loaded NeoHDL, which points to uptake by the asialoglycoprotein receptor. Subcellular fractionation of the liver indicated that lactosylated [3H]IDU-Ol2-loaded NeoHDL does not merely associate to cells, but is internalized and delivered to the lysosomes. In conclusion, we show that IDU can be specifically targeted to the parenchymal liver cell. Conversion of the water-soluble parent drug into a lipophilic prodrug that is incorporated into a lactosylated reconstituted HDL particle, is an approach that may also be used to deliver other water-soluble drugs to the parenchymal liver cells. This may lead to more effective therapy for liver diseases such as hepatitis B.

Selective induction of cytochrome P450 3A1 by dexamethasone in cultured rat hepatocytes: analysis with a novel reverse transcriptase-polymerase chain reaction assay section sign.

The study of drug metabolism in cultured rat hepatocytes is hampered by the rapid loss of the expression of cytochrome P450 enzymes. Nevertheless, the activity of cytochrome P450 3A (CYP3A), one of the most important isoenzymes for drug metabolism, can be elevated by chemical inducers. In the present study, we investigated in cultured rat hepatocytes the induction of all four currently identified CYP3A isoforms by dexamethasone, and compared the results obtained in vitro with the induction profile of dexamethasone in vivo. To this end, CYP3A mRNA levels were quantified with a novel, radioactive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and CYP3A enzymatic activity was measured by a testosterone hydroxylation assay. In the RT-PCR assay, CYP3A isoforms were co-amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of radioactively labeled nucleotides. This resulted in an extremely sensitive and accurate determination of CYP3A expression levels, relative to those of GAPDH. Using this RT-PCR assay, it was found that the expression of all CYP3A isoforms in rat hepatocytes, cultured on a collagen matrix, was decreased by 80-90% within one day of cultivation. After addition of dexamethasone, at one day after isolation, CYP3A1 mRNA levels were elevated to levels comparable to those in freshly isolated hepatocytes within two days. In contrast, CYP3A2, CYP3A9, and CYP3A18 mRNA levels were not affected by dexamethasone treatment, and were hardly detectable after three days of cultivation. CYP3A enzymatic activity was also induced in cultured hepatocytes (approximately 6-fold) after addition of dexamethasone. In vivo, CYP3A1 mRNA levels increased 45-fold after dexamethasone administration. However, in contrast to the situation in cultured hepatocytes, CYP3A2 and CYP3A18 were also induced, albeit to a lesser extent (4- and 7-fold elevated mRNA levels, respectively). We conclude that the selective induction of CYP3A1 in dexamethasone-treated rat hepatocytes allows the study of biotransformation reactions by CYP3A1, without interference by any of the other CYP3A isoenzymes.

Modification of the plasma clearance and liver uptake of steroid ester-conjugated oligodeoxynucleotides by association with (lactosylated) low-density lipoprotein.

Low-density lipoprotein (LDL) has been proposed as carrier for the selective delivery of anticancer drugs to tumor cells. We reported earlier the association of several lipidic steroid-conjugated anticancer oligodeoxynucleotides (ODNs) with LDL. In the present study, we determined the stability of these complexes. When the complexes were incubated with a mixture of high-density lipoprotein and albumin, or with rat plasma, the oleoyl steroid-conjugated ODNs appeared to be more stably associated with LDL than the cholesteryl-conjugated ODN. Intravenously injected free lipid-ODNs were very rapidly cleared from the circulation of rats. The area under the curve (AUC) of the lipid-ODNs in plasma was <0.4 microg x min/mL. After complexation with LDL, plasma clearance of the lipid-ODNs was delayed. This was most evident for ODN-5, the ODN conjugated with the oleoyl ester of lithocholic acid (AUC = 6.82 +/- 1.34 microg x min/mL). The AUC of ODN-4, a cholesteryl-conjugated ODN, was 1.49 +/- 0.37 microg x min/mL. In addition, the liver uptake of the LDL-complexed lipid-ODNs was reduced. The lipid-ODNs were also administered as a complex with lactosylated LDL, a modified LDL particle that is selectively taken up by the liver. A high proportion of ODN-5 was transported to the liver along with lactosylated LDL (69.1 +/- 8.1% of the dose at 15 min after injection), whereas much less ODN-4 was transported (36.6 +/- 0.1% of the dose at 15 min after injection). We conclude that the oleoyl ester of lithocholic acid is a more potent lipid anchor than the other steroid lipid anchors. Because of the stable association, the oleoyl ester of lithocholic acid is an interesting candidate for tumor targeting of anticancer ODNs with lipoproteins.

Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein.

The efficacy of antisense oligonucleotides depends on the ability to reach in vivo their target cells. We aim to develop strategies to enhance uptake of phosphorothioate oligodeoxynucleotides by Kupffer cells. To this end, we conjugated cholesterol to ISIS-3082, a phosphorothioate oligodeoxynucleotide specific for intercellular adhesion molecule-1. The cholesterol-conjugated oligonucleotide, denoted ISIS-9388, associated readily with lactosylated low-density lipoprotein (LacLDL), a lipidic carrier that is taken up by galactose receptors on Kupffer cells. Association of up to 10 molecules of ISIS-9388 per LacLDL particle did not induce aggregation. LacLDL-associated [3H]ISIS-9388 was rapidly taken up by the liver after injection into rats (52.9+/-1.8% of the dose within 2 min versus 18.6+/-2.8% for ISIS-3082). N-acetylgalactosamine inhibited hepatic uptake, indicating involvement of galactose-specific receptors. Liver cells were isolated at 60 min after injection of LacLDL-associated [3H]ISIS-9388. Kupffer cells displayed the highest uptake: 88.1+/-24.7 ng of oligonucleotide/mg of cell protein, which is 6-14 times higher than after injection of free ISIS-9388 or ISIS-3082 (15.0+/-3.8 ng and 6.3+/-1.4 ng, respectively). It can be calculated that Kupffer cells contribute 43.9+/-5.4% to the liver uptake (free ISIS-9388 and ISIS-3083 14.5+/-3.1% and 8.3+/-3.2%, respectively). In conclusion, conjugation of a phosphorothioate oligodeoxynucleotide with cholesterol and its subsequent association with LacLDL results in a substantially increased Kupffer cell uptake of the oligonucleotide. As Kupffer cells play a key role in inflammation, our approach may be utilized to improve antisense-based therapeutic intervention during inflammation.

Interactions of lipid-oligonucleotide conjugates with low-density lipoprotein.

The ability of antisense oligonucleotides to interdict, sequence-specifically, the expression of pathogenic genes affords an exciting new strategy for therapeutic intervention (1-3). Oligonucleotides with physiological phosphodiester internucleotide bonds are rapidly degraded, predominantly by exonucleases. Numerous oligonucleotide analogs have therefore been synthesized to confer resistance toward nuclease activity (3). The phosphorothioate analog is the most extensively studied, and phosphorothioate oligodeoxynucleotides have been shown to be potent inhibitors of the expression of their target genes in vitro and in vivo (1,3). However, phosphorothioate oligodeoxynucleotides also bind avidly and nonspecifically to proteins, thus provoking a variety of non-antisense effects (4). Oligonucleotide analogs that do not bind to proteins are therefore expected to display less nonantisense side effects. However, protein binding also affects the in vivo disposition of oligonucleotides. Nonphosphorothioate oligonucelotide analogs generally do not bind to serum proteins, and are therefore rapidly cleared from the circulation, protein-bound phosphorothioate oligodeoxynucelotides circulate much longer (5,6).

Concanavalin A induces Ca2+ mobilization, but only minimal inositol phospholipid breakdown in mouse B cells.

Concanavalin A (Con A) induces DNA synthesis in bulk cultures of murine T cells. In contrast, the lectin has been shown to stimulate purified B cells to leave G0, but not to proliferate. We demonstrate here that Con A induces comparable increases in intracellular Ca2+ levels in the two cell types. In B cells this appears to be due to Ca2+ influx. As expected, the lectin also provokes significant degradation of inositol phospholipids in T cells. However, it causes only minimal release of inositol phosphates in B cells. We therefore postulate that Con A may stimulate B cells by causing influx of Ca2+. In line with this, activation of B cells by the lectin is inhibited by cyclosporine, which preferentially affects stimulation of lymphocytes by Ca2+ mobilizing agents.

Cyclosporine does not inhibit mitogen-induced inositol phospholipid degradation in mouse lymphocytes.

The degradation of phosphatidylinositol bisphosphate (PIP2) to diacylglycerol and inositol trisphosphate is elicited by ligand-receptor interactions in many cell types, and may be involved in the induction of cell growth. The mitogens concanavalin A and anti-immunoglobulin antibodies have previously been shown to induce degradation of PIP2 in mouse thymocytes and B cells, respectively. We have now investigated the effects of the immunosuppressive peptide cyclosporine (CS) on this response, since CS appears to inhibit an early step in lymphocyte activation by mitogens that induce PIP2 degradation and Ca2+ mobilization. We found that CS, at doses that completely abrogated the proliferative responses, did not affect the degradation of inositol phospholipids in either thymocytes or B cells. We therefore conclude that if PIP2 degradation is implicated in lymphocyte activation, then CS does not interfere with the second messenger production initiated by PIP2 breakdown, but rather with a later event(s) elicited by this pathway.

Tumor-promoting phorbol esters suppress receptor-stimulated inositol phospholipid degradation and Ca2+ mobilization in mouse lymphocytes.

Anti-immunoglobulin antibodies (anti-Ig) provoke the rapid breakdown of phosphatidylinositol bisphosphate (PIP2), elevation of cytoplasmic Ca2+ levels ([Ca2+]i) and activation of protein kinase C (PKC) in B lymphocytes. Tumor-promoting phorbol esters, like phorbol myristate acetate, also activate PKC, but inhibit anti-Ig-induced B cell proliferation. To investigate the basis of the latter effect, we studied the influence of phorbol esters on PIP2 degradation and [Ca2+]i in murine B cells. The results show that PKC-activating phorbol esters cause marked inhibition of anti-Ig-stimulated PIP2 breakdown and Ca2+ mobilization. In addition, these agents inhibit concanavalin A-provoked Ca2+ influx, lower resting cytoplasmic Ca2+ levels and reduce ionophore-induced Ca2+ influx in B cells. Apparently, PKC stimulation causes feedback inhibition of receptor signalling, not only by suppressing PIP2 degradation, but also by exerting additional complex effects on the control of [Ca2+]i in B cells. It is, however, not clear how these findings relate to the anti-proliferative effects of phorbol esters on B cells.

Lactosylated high density lipoprotein: a potential carrier for the site-specific delivery of drugs to parenchymal liver cells.

Mammalian liver contains two types of galactose receptors, specific for Kupffer or parenchymal cells. Because galactose-specific receptors are largely confined to the liver, galactose-bearing carriers are promising vehicles for the specific delivery of drugs to liver cells. In the present study, high density lipoprotein (HDL), a spherical particle with a diameter of 10 nm, in which a variety of lipophilic drugs can be incorporated, was provided with galactose residues by reductive lactosamination. After injection into rats, lactosylated 125I-HDL was rapidly cleared from the plasma (half-life, less than 1 min). Ten minutes after injection, the liver contained about 95% of the dose, whereas only small amounts of radioactivity were found in other tissues. The hepatic uptake was inhibited by preinjection with N-acetylgalactosamine, which indicates that the hepatic recognition sites are galactose specific. Subcellular fractionation of the liver indicated that recognition of lactosylated HDL is followed by internalization and degradation of the apoprotein in the lysosomes. Liver cells were isolated at 10 min after injection of lactosylated 125I-HDL, and it was found that uptake occurs almost exclusively by parenchymal cells. These cells contained about 98% of the hepatic radioactivity. The liver uptake of the lipid moiety of lactosylated HDL, labeled with [3H]cholesteryl oleate, was identical to that of the 125I-labeled apoproteins, which indicates that the particle is taken up as a unit. Thus, lactosylated HDL is taken up rapidly and selectively by parenchymal liver cells, and it appears that it might be a very effective vehicle for the specific delivery of lipophilic drugs to these cells.

Synthesis of the dioleoyl derivative of iododeoxyuridine and its incorporation into reconstituted high density lipoprotein particles.

We investigated the potential use of reconstituted HDL particles (NeoHDL) as a carrier for lipophilic (pro)drugs. The antiviral drug iododeoxyuridine (IDU) was used as model compound. [3H]-IDU was derivatized with two oleoyl residues to dioleoyl[3H]iododeoxyuridine ([3H]IDU-Ol2), and the lipophilic prodrug was incorporated into NeoHDL by cosonication of [3H]IDU-Ol2 with lipids and HDL apoproteins. NeoHDL particles with the same density, size, and electrophoretic mobility as native HDL were obtained, which contained 7.3 +/- 0.8% (w/w) [3H]IDU-Ol2 (about 30 molecules of prodrug per particle). NeoHDL-associated [3H]IDU-Ol2 was stable during 2 h of incubation with human plasma; the prodrug was not appreciably hydrolyzed, nor exchanged with LDL. After intravenous injection of [3H]-IDU-Ol2-loaded 125I-NeoHDL into rats, [3H]IDU-Ol2 disappeared more rapidly from the circulation than the 125I-apoproteins (78.0 +/- 8.0% vs 30.1 +/- 4.5% of the dose cleared from plasma in 60 min, respectively). The hepatic association of the prodrug was higher than that of the apoproteins (21.6 +/- 0.5 vs 5.2 +/- 1.0% of the dose at 10 min after injection, respectively). As selective clearance and uptake of lipid esters is also observed with native HDL, this suggests that, in vivo, prodrug-loaded NeoHDL may be subject to physiological HDL-specific processing. Lactosylated [3H]IDU-Ol2-loaded 125I-NeoHDL, which contains galactose residues that can be recognized by galactose receptors on parenchymal liver cells, was rapidly cleared from plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: analysis with indo-1.

The new Ca2+-probe indo-1 has a high fluorescence intensity, which allows low intracellular dye loadings. Stimulation of indo-1-loaded mouse B cells with anti-Ig antibodies provoked rapid rise of free cytoplasmic Ca2+ from 100 nM to greater than 1 microM, followed by a decline to a plateau at 300-400 nM. The initial rapid rise was not detected in quin2-loaded cells, presumably due to the Ca2+-buffering effects of the dye. The sustained Ca2+ increase was due to influx, whereas the initial rise was caused by release from intracellular stores. The magnitudes of Ca2+ release and inositol trisphosphate release were closely correlated. Concanavalin A does not provoke inositol trisphosphate release in mouse B cells. It did not induce a rapid initial Ca2+ rise in indo-1-loaded B cells either, but only a sustained increase to 200-300 nM. Finally, Ca2+ influx induced by both anti-Ig and concanavalin A were not affected by membrane depolarization.

Activation and proliferation signals in mouse B cells. V.A. comparison of the effects of intact (IgG) and F (ab')2 anti-mu or anti-delta antibodies.

This study compares the effects of soluble intact (IgG) and F(ab')2 rabbit anti-mu and anti-delta antibodies on mouse B cells. The results show that while the F(ab')2 antibodies to both isotypes induce polyclonal B cell proliferation, the IgG antibodies are not mitogenic, but rather inhibit DNA synthesis induced by the homologous F(ab')2 fragments. Furthermore, intact anti-mu antibodies inhibit mitogenesis induced by F(ab')2 anti-delta, and vice versa. However, the intact antibodies to both isotypes promote early changes characteristic of B-cell activation, namely, increased Ia antigen expression, and priming for a facilitated proliferative response to F(ab')2 anti-Ig. In addition, F(ab')2 anti-mu and anti-delta both induce the breakdown of phosphatidylinositol phospholipids in B cells, an early consequence of Ig receptor cross-linking which may be involved in the induction of cell growth. These results therefore indicate that stimulating mature B cells via IgM or IgD receptors produces indistinguishable early effects, and that cross-linking Fc and surface Ig receptors by intact anti-Ig generates a dominant inhibitory signal, regardless of which isotype is involved.