Acquisition of mitogenic responsiveness by nonresponding lymphocytes upon insertion of appropriate membrane components.

Jakobovits, Aya; Sharon, Nathan; Zan‐Bar, Israel

doi:10.1084/jem.156.4.1274

Cited by 44 publications

(21 citation statements)

References 13 publications

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“…The Lps gene may regulate the expression of some membrane component that recognizes LPS and there is some evidence to support this hypothesis. Studies using the cell fusion technique have shown that the expression of a genetic defect in C3H/HeJ mice is located on the membrane fraction and not in the nucleus (10,30). However, a number of studies on the binding of LPS to cells have not shown a difference between LPS-responsiveness and nonresponsiveness.…”

Section: Discussionmentioning

confidence: 99%

Identification of Re Lipopolysaccharide‐Binding Protein on Murine Erythrocyte Membrane

Kirikae

Inada

Hirata

et al. 1988

Microbiology and Immunology

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Our recent studies have suggested that bacterial lipopolysaccharide (LPS) attaches to Pronase-sensitive proteins on the murine erythrocyte membrane. In the present study, in order to identify the LPS-binding protein on the murine erythrocyte membrane, a unique method to detect LPS-binding protein on a nitrocellulose membrane was developed. Murine erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then transferred electrophoretically onto a nitrocellulose membrane. The membrane was incubated with LPS of Salmonella minnesota R595 (Re LPS) in phosphate-buffered saline (PBS), after the remaining sites were blocked with gelatin in PBS. We were able to obtain a non-background stain by adding the nonionic detergent octylglucoside at the low concentration of 0.1 % to the Re LPS solution. The Re LPS bound to the protein on the nitrocellulose membrane was exposed to affinity purified anti-Re LPS antibodies (IgG) and then to alkaline phosphatase-conjugated anti-IgG. The alkaline phosphatase was detected on the membrane by an enzymatic reaction. This method demonstrated that Re LPS was bound to an erythrocyte protein of 96 kDa. Treatment of erythrocytes with Pronase led to disappearance of the Re LPS-binding protein on the erythrocyte membrane. There was no difference between LPSresponder and LPS-nonresponder murine erythrocyte membranes in amount and molecular weight of the Re LPS-binding protein.Lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria and its biologically active moiety, lipid A, induces a variety of pathophysiologic effects in animals. Recent approaches by both analysis and chemical synthesis of lipid A are elucidating the structural requirement for biological activities of LPS (9,14,22). However, the mechanism by which LPS activates host cells, especially the existence of a specific receptor for LPS on the cells, remains open to question.For the purpose of clarifying the interaction between LPS and cell membrane, we have used erythrocytes as a model of the cell membrane. Thus, we have found that LPS, especially R-form LPS, and lipid A directly agglutinates erythrocytes from some kinds of animals such as mice and rabbits (12). In this direct hemagglutina-33

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Section: Discussionmentioning

confidence: 99%

Identification of Re Lipopolysaccharide‐Binding Protein on Murine Erythrocyte Membrane

Kirikae

Inada

Hirata

et al. 1988

Microbiology and Immunology

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“…We used the method described by Jakobovits et al [27] to insert PHA receptors into purified B lymphocytes. Vesicles made of PC/PS were prepared by dialysis of a detergent (DTAB) solution containing affinity-purified porcine PHA receptors and Sendai virus fusogenic HN and F proteins.…”

Section: Assay Of Reconstituted B Lymphocytesmentioning

confidence: 99%

B Lymphocytes Respond Specifically to Phytohaemagglutinin after Liposome‐Dependent Transfer of Purified Phytohaemagglutinin Receptors

Letellier

Dupuis

1987

Scand J Immunol

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Phytohaemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography from porcine splenic lymphocytes, were reconstituted into vesicles made of phosphatidylcholine and phosphatidylserine, concomitantly with Sendai virus fusogenic proteins HN and F. The vesicles were used as a vehicle to insert the PHA receptor glycoproteins into a highly enriched population of porcine B lymphocytes. Fluorescence analyses showed that 52 +/- 2% of the reconstituted B cells had incorporated the lectin receptors. The modified B lymphocytes were assayed for their response (tritiated thymidine incorporation into nucleic acids) to PHA, concanavalin A (Con A), or to lipopolysaccharide (LPS). The results showed that porcine B cells fused with vesicles containing only viral fusogenic proteins failed to respond to either PHA or Con A. Tritiated thymidine incorporation was similar to background values. The cells did, however, respond to LPS with values of label incorporation similar to those observed in the case of pre-fused B lymphocytes. When purified B lymphocytes were fused with vesicles containing PHA receptors and viral fusogenic proteins, assays of thymidine incorporation showed a statistically significant (P less than 0.001) and specific response of the modified cells to PHA stimulation. Reconstituted cells cultured in the presence of PHA incorporated approximately nine times more radioactive label than pre-fused cells or cells fused with vesicles containing only fusogenic viral proteins. In marked contrast, reconstituted B lymphocytes did not show any significant label incorporation above background level in response to Con A, but they retained their ability to respond to LPS. Our findings suggest that B lymphocytes can be made to respond specifically to PHA by insertion of appropriate lymphocyte-derived receptors.

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“…A rabbit serum raised against LPS-responsive B cells, and absorbed with C3H/HeJ cells, was able to compete with endotoxin for binding on LPS-responsive B cells, and to inhibit their mitogenic response [6]. Furthermore, Jakobovits et al [7] were able partially to reconstitute LPS responsiveness in C3H/HeJ B lymphocytes by fusing them with plasma membranes from syngeneic LPS-responder B cells. These data suggested the existence of a cell-surface signaling receptor.…”

Section: Introductionmentioning

confidence: 99%

Structural features involved in the mitogenic activity of bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice

Lasfargues

1993

FEMS Immunology and Medical Microbiology

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Spleen cells from the C3H/HeJ mouse strain cannot be stimulated by many smooth-type lipopolysaccharides (LPSs), and by the main biologically-active region (lipid A) of these molecules. The genetic origin of this defect (expression of the mutant allele Lpsd at the chromosome 4 locus) was established over 20 years ago, but its biochemical nature has remained undefined. Several investigators have noted, however, that some particular LPSs can bypass this defect, and stimulate the proliferation of C3H/HeJ B lymphocytes. In this study we compare the mitogenic activities of the LPSs isolated from a wild strain (1414) and from a mutant 'rough' strain (A100) of Bordetella pertussis. Both LPS-1414 and LPS-A100 were mitogenic for C3H/HeJ spleen cells, but their lipid A fragments were not. This indicates that a carbohydrate structure proximal to lipid A is involved in the mitogenic activity. However, the isolated polysaccharides were not mitogenic. Four sugars are common to both LPS-1414 and LPS-A100: an heptose, and three sugars bearing free amino groups. After removal of these four sugars from the LPSs by nitrous acid treatment, the recovered lipooligosaccharides were not mitogenic in Lpsd spleen cells. The results suggest that substructures present in lipid A and in this group of four sugars are both required for induction of a mitogenic effect in Lpsd splenocytes, whereas lipid A alone can stimulate Lpsn spleen cells.

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Acquisition of mitogenic responsiveness by nonresponding lymphocytes upon insertion of appropriate membrane components.

Cited by 44 publications

References 13 publications

Identification of Re Lipopolysaccharide‐Binding Protein on Murine Erythrocyte Membrane

Identification of Re Lipopolysaccharide‐Binding Protein on Murine Erythrocyte Membrane

B Lymphocytes Respond Specifically to Phytohaemagglutinin after Liposome‐Dependent Transfer of Purified Phytohaemagglutinin Receptors

Structural features involved in the mitogenic activity of bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice

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