Pre-B-cell development in the absence of lambda 5 in transgenic mice expressing a heavy-chain disease protein.

BACKGROUND: Heavy-chain diseases (HCDs) are human lymphoproliferative neoplasias that are characterized by the secretion of truncated immunoglobulin heavy chains devoid of light chains. We have previously proposed--by analogy to the process by which mutated growth factor receptors can be oncogenic--that because the genetic defects in HCDs result in the production of abnormal membrane-associated heavy chains lacking an antigen-binding domain, these abnormal B-cell antigen receptors might engage in ligand-independent signalling. Normal pre-B-cell development requires the presence of the pre-B-cell receptor, formed by the association of mu heavy chains with two polypeptides--so-called surrogate light chains, Vpre-B and lambda 5--that are homologous to the variable and constant portions of immunoglobulin light chains, respectively. To assess whether amino-terminal truncation of membrane-associated heavy chains results in their constitutive activation, we have examined the ability of a HCD-associated mu protein to promote pre-B-cell development in transgenic mice. RESULTS: When the mu HCD transgene is introduced into SCID mice, CD43- pre-B cells develop normally. To determine whether this pre-B-cell development requires surrogate light chains, we backcrossed mice expressing full-length or truncated mu transgenes with lambda 5-deficient mice. Our results show that the truncated heavy chain, but not the normal chain, is able to promote pre-B-cell development in the absence of lambda 5. We also show that truncated mu chains spontaneously aggregate at the surface of bone marrow cells. CONCLUSIONS: Expression of the truncated mu heavy chain overrides a tightly controlled step of pre-B-cell development, which strongly suggests that a constitutive signal is delivered by the truncated mu chain disease protein. The self-aggregation of mu chain disease proteins might account for this constitutive activation. We conclude that amino-terminal truncation of heavy chains could play a role in the genesis of HCD neoplasia if it occurs at an appropriate stage of B-cell differentiation, namely in a mature B cell.

Apical and basal Forssman antigen in MDCK II cells: a morphological and quantitative study.

We have studied the surface distribution of a glycosphingolipid (the Forssman antigen) in MDCK II and CCL39 cells. The Forssman antigen is mobile on the surface of both these cell lines. Its surface distribution is homogenous on non-polarized cells. Under conditions where MDCK II cells are well polarized, the Forssman antigen is present in equal amounts on the apical membrane and on the basal membrane and its processes. Very little Forssman antigen can be detected on the lateral membrane. The nature of the mechanism excluding the Forssman antigen from the lateral domain remains to be determined. This surface distribution is established within hours after plating and was observed with cells grown on different types of filters. The surface density of the Forssman antigen on the apical and on the basal domain has been estimated. No involvement of the basal Forssman antigen in cell attachment could be demonstrated. However, the apical Forssman antigen appears to be essential to the establishment of the cells in culture.

Differential distribution of galactosylceramide, H antigen, and carcinoembryonic antigen in rhesus macaque digestive mucosa.

The most common route of transmission of HIV is via the mucosa. We compared human and macaque intestinal epithelia to determine whether the SIV macaque system can be used as a model to study HIV transmission by the rectal route. The overall morphology of the macaque gut mucosa is very similar to that of humans. Differentiation markers follow the same pattern as in the human system. The carcinoembryonic antigen (CEA) is apical in epithelial cells of the rectum and is absent from the small intestine. Blood group H antigen is expressed by enterocytes but not by colonocytes or rectocytes. Galactosylceramide, a potential alternative receptor for HIV in epithelial cells, is expressed in all intestinal segments as in humans. In absorptive cells it is apical and intracellular in the rectum, colon, and cecum, whereas it is only intracellular in small intestine. In goblet cells the galactosylceramide is present in intracellular vacuoles in all segments. It is also present on the membrane of mucous granules in colon and small intestine but not in rectum. We therefore believe that the macaque digestive tract may constitute a good model for the human digestive tract in the transmission of lentiviruses.

Chimpanzee CXCR4 and CCR5 act as coreceptors for HIV type 1.

Chemokine receptors are molecules involved in the fusion of immunodeficiency viruses after their attachment. As chimpanzees are the animal model for infection by HIV-1, we cloned and sequenced chimpanzee CXCR4 and CCR5 from PBMCs. Chimpanzee CXCR4 was found to be identical to human CXCR4, which provides an explanation for the sensitivity of chimpanzees to lymphotropic isolates of HIV-1. Chimpanzee CCR5 showed two substitutions with respect to human CCR5. However, we show that the macrophage-tropic isolate HIV-1-Ba-L can use chimpanzee CCR5 as a fusion receptor. Therefore, the resistance of chimpanzee PBMCs to infection by macrophage-tropic isolates of HIV-1 is unlikely to be due to substitutions in CCR5.

Apical to basolateral surface area ratio and polarity of MDCK cells grown on different supports.

We have established conditions under which Madin-Darby canine kidney cells develop a well-polarized monolayer on polycarbonate filters and on transparent filters. These filters have biochemical and mechanical advantages over the nitrocellulose filters which have been widely used. Transepithelial resistance was established 10 h after plating and stabilized after 24 h. The distribution of protein antigens was followed by surface immunofluorescence and quantitated by a surface immunoassay that we developed. Uvomorulin was localized to the lateral membrane, with low amounts detectable on the basal membrane. The 58-kDa antigen was distributed over the entire basolateral domain, including cell processes extending into the filter pores. This distribution was confirmed by immunogold labeling of frozen sections. The 114-kDa antigen was found to be present at similar surface densities on both the apical and the basolateral domain. The support used for growth had profound effects on the cell morphology. A morphometric analysis of the plasma membrane of both strains of the cell line showed an increase in the number and size of the microvilli, and a smoother basal membrane as compared to published data on nitrocellulose filters. The apical to basolateral surface area ratio was therefore modified.

High level O-acetylation of sialic acids on N-linked oligosaccharides of rat liver membranes. Differential subcellular distribution of 7- and 9-O-acetyl groups and of enzymes involved in their regulation.

O-Acetylation of sialic acids has previously been considered an uncommon modification found on certain salivary mucins and neural gangliosides. We show here that glycosidically bound sialic acids from total membranes of rat liver have surprisingly high levels (approximately 20%) of O-acetylation at the 7- or 9-position. This O-acetylation is further enriched in N-linked oligosaccharides but is barely detectable in ganglioside fractions from the same tissue. The position of O-acetylation on the sialic acid side chain varies between different subcellular fractions. In particular, 7-O-acetylation was enriched in lysosomal membranes and 9-O-acetylation in plasma membranes, whereas Golgi membranes contained both types. This distribution fits with the ability of the rat liver sialate: O-acetyltransferase(s) to synthesize both 7- and 9-O-acetyl esters (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426) and the fact that 7-O-acetyl esters can migrate to the 9-position at physiological temperature but only under neutral or mildly alkaline conditions. Subcellular fractionation shows that sialate:O-acetyltransferase activity directed toward endogenous acceptors is enriched in Golgi fractions, whereas an intralumenal sialic acid-specific O-acetylesterase activity is not. The O-acetyltransferase is labile and difficult to solubilize in the intact state and cannot be assayed with exogenous acceptors. However, a prelabeled [3H]acetyl intermediate can be solubilized from Golgi membranes with Triton X-100 and is stable for a prolonged time in the cold. In contrast to the transferase, the lumenal esterase is easily released in a stable and water-soluble form from membrane fractions by saponin permeabilization or repeated freeze-thaw. In keeping with this finding, differential subcellular fractionation and continuous sucrose gradients indicate that this enzyme is enriched in lysosomal fractions (see also the accompanying paper (Butor, C., Higa, H. H., and Varki, A. (1993) J. Biol. Chem. 268, 10207-10213). Based upon findings reported in this and previous studies, a model is proposed for the biosynthesis, maturation, and turnover of 7- and 9-O-acetyl esters on the sialic acids of N-linked oligosaccharides that are attached to membrane-bound proteins in the rat liver.

Structural, immunological, and biosynthetic studies of a sialic acid-specific O-acetylesterase from rat liver.

We have previously described a membrane-associated intralumenal sialic acid-specific 9-O-acetylesterase (LSE) from rat liver (Higa, H. H., Manzi, A., and Varki, A. (1989) J. Biol. Chem. 264, 19435-19442). Unlike a cytosolic sialate: O-acetylesterase (CSE) with similar specificity, the LSE carries N-linked oligosaccharides. A polyclonal monospecific antibody against homogenous LSE does not cross-react with the CSE. Monoclonal antibodies distinguish between the LSE and another N-glycosylated esterase that tends to partially co-purify with it. Amino-terminal sequencing of the LSE subunits indicates that it is distinct from previously described esterases and shows no homology to any other known proteins. In contrast, the esterase that partially co-purifies is similar but not identical to previously described "microsomal" esterases from rat liver. The LSE is also expressed in several hepatoma cell lines. Pulse-chase studies indicate that the two LSE subunits arise from a single precursor of approximately 65 kDa which yields a core polypeptide of apparent molecular mass approximately 53 kDa upon deglycosylation with peptide: N-glycosidase F. The protein quickly becomes partly resistant to endo-beta-N-acetylglucosaminidase H but remains sensitive to peptide: N-glycosidase F, indicating N-linked oligosaccharide processing during passage through the Golgi. After several hours, the precursor undergoes proteolysis, generating the mature heterodimeric protein of approximately 58 kDa, with subunits of approximately 38 and approximately 28 kDa. A portion of newly synthesized LSE is secreted into the medium intact, indicating that the cleavage normally takes place after diversion from the secretory pathway. These temporal changes and precursor-product distribution are reminiscent of some lysosomal acid hydrolases. In fact, immunofluorescence studies and Triton WR-1339 shift experiments suggest a lysosomal localization for this enzyme. Additional evidence for this, and the role of the LSE in O-acetylated sialic acid turnover are discussed in the accompanying paper (Butor, C., Diaz, S., and Varki, A. (1993) J. Biol. Chem. 268, 10197-10206).

O-acetylation and de-O-acetylation of sialic acids. O-acetylation of sialic acids in the rat liver Golgi apparatus involves an acetyl intermediate and essential histidine and lysine residues--a transmembrane reaction?

Isolated intact rat liver Golgi vesicles utilize [acetyl-3H]coenzyme A to add 3H-O-acetyl esters to sialic acids of internally facing endogenous glycoproteins. During this reaction, [3H]acetate also accumulates in the vesicles, even though the vesicles are impermeant to free acetate. On the other hand, entry of intact AcCoA into the lumen of the vesicles could not be demonstrated, and permeabilization of the vesicles did not alter the reaction substantially (Diaz, S., Higa, H. H., Hayes, B. K., and Varki, A. (1989) J. Biol. Chem. 264, 19416-19426). When vesicles prelabeled with [acetyl-3H] coenzyme A are permeabilized with saponin, we can demonstrate a [3H]acetyl intermediate in the membrane that can transfer label to the 7- and 9-positions of exogenously added free N-acetylneuraminic acid but not to glucuronic acid or CMP-N-acetylneuraminic acid. This labeled acetyl intermediate represents a significant portion of the radioactivity incorporated into the membranes during the initial incubation and cannot be accounted for by nonspecifically "trapped" acetyl-CoA in the permeabilized vesicles. There was no evidence for involvement of acetylcarnitine or acetyl phosphate as an intermediate. The overall acetylation reaction appears to involve two steps. The first step (utilization of exogenous acetyl-CoA to form the acetyl intermediate) is inhibited by coenzyme A-SH (apparent Ki = 24-29 microM), whereas the second (transfer from the acetyl intermediate to sialic acid) is not affected by millimolar concentrations of the nucleotide. Studies with amino acid-modifying reagents indicate that 1 or more histidine residues are involved in the first step of the acetylation reaction. Diethylpyrocarbonate (which can react with both nonsubstituted and singly acetylated histidine residues) also blocks the second reaction, indicating that the acetyl intermediate on both sides of the membrane involves histidine residue(s). Taken together with data presented in the preceding paper, these results indicate that the acetylation of sialic acids in Golgi vesicles may occur by a transmembrane reaction, similar to that described for the acetylation of glucosamine in lysosomes (Bame, K. J., and Rome, L. H. (1985) J. Biol. Chem. 260, 11293-11299). However, several features of this Golgi reaction distinguish it from the lysosomal one, including the nature and kinetics of the reaction and the additional involvement of an essential lysine residue. The accumulation of free acetate in the lumen of the vesicles during the reaction may occur by abortive acetylation (viz. transfer of label from the acetyl intermediate to water). It is not clear if this is an artifact that occurs only in the in vitro reaction.

Co-localization of hydrolytic enzymes with widely disparate pH optima: implications for the regulation of lysosomal pH.

Lysosomes are traditionally defined by their acidic interior, their content of degradative 'acid hydrolases', and the presence of distinctive membrane proteins. Terminal degradation of the N-linked oligosaccharides of glycoproteins takes place in lysosomes, and involves several hydrolases, many of which are known to have acidic pH optima. However, a sialic acid-specific 9-O-acetyl-esterase and a glycosyl-N-asparaginase, which degrade the outer- and inner-most linkages of N-linked oligosaccharides, respectively, both have pH optima in the neutral to alkaline range. By immunoelectron microscopy, these enzymes co-localize in lysosomes with several conventional acid hydrolases and with lysosomal membrane glycoproteins. Factors modifying the pH/activity profiles of these enzymes could not be found in lysosomal extracts. Thus, the function of the enzymes with neutral pH optima must depend either upon their minimal residual activity at acidic pH, or upon the possibility that lysosomes are not always strongly acidic. Indeed, when lysosomes are marked in living cells by uptake of fluorescently labeled mannose 6-phosphorylated proteins, the labeled organelles do not all rapidly accumulate Acridine Orange, a vital stain that is specific for acidic compartments. One plausible explanation is that lysosomal pH fluctuates, allowing hydrolytic enzymes with a wide range of pH optima to efficiently degrade macromolecules.

Solid phase cytometry for detection of rare events.

We describe a simple and rapid laser scanning device for solid phase cytometry. This system can detect and count fluorescent cells over a 22 mm diameter surface (membrane or glass circle) as well as quantify the fluorescence that they emit. A comprehensive discrimination package includes optical and software parameters and accurately distinguishes between valid signals (e.g., labelled cells) and nonspecific signals (e.g., auto-fluorescent particles and debris). Any event detected may also be easily confirmed by visual observation after transfer of the sample to an epifluorescence microscope fitted with a motorized stage driven by the system. We show a linear relationship between the amount of fluorescein coupled to the cells and the fluorescence signal of the cells detected. This approach is not destructive and further characterization of the sample may be carried out. We have been able to detect rare cellular events at a frequency of 10(-7) in 3 min. Potential applications include monitoring of residual disease in oncology and detection of virus-infected cells circulating at very low frequencies.

Dissemination of SIV after rectal infection preferentially involves paracolic germinal centers.

Homosexual transmission remains a major mode of contamination in developed countries. Early virological and immunological events in lymphoid tissues are known to be important for the outcome of HIV infections. Little data are available, however, on viral dissemination during primary rectal infection. We therefore studied this aspect of rectal infection in rhesus macaques inoculated with the biological isolate SIVmac251. We show that infection is established initially in lymph nodes draining the rectum. Infected cells and virions are localized mainly in germinal centers at that stage. With increasing viral burden, infected cells are found throughout the lymph node parenchyma. In addition the difference in viral load between lymph nodes draining the rectum and other lymph nodes is attenuated or abolished. We discuss this pattern of viral dissemination with respect to the physiology of the mucosal immune system. The pattern and kinetics of viral dissemination after rectal infection have important implications for the development of efficient mucosal vaccines.

Solid phase cytometry allows rapid in situ quantification of human papilloma virus infection in biopsy material.

Solid phase cytometry would be an asset for many histological and cytological studies. Current microscope-based cytometers and image analysis systems are too slow to analyze specimens several millimeters wide. We have recently shown that a rapid wide area laser scanning device that operates on solid supports has a linear response. We assess it here for solid phase cytometry. Each cell detected by the cytometer can be automatically positioned for visual observation in the field of an epifluorescence microscope (conventional or confocal) in which the stage is driven by the instrument's computer. We were able to detect and map human papillomavirus-infected cells labeled by fluorescent in situ hybridization in cervical condyloma biopsies. We could quantify the fluorescence emitted by these cells and show differences of up to 35-fold in fluorescence intensity between individual cells. These differences in intensity might reflect differences in viral copy number. The potential of the system to provide fast, reliable and reproducible analyses of solid tissue samples is discussed.