WEHI-3 Cells Research Articles

Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences

Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) >> h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.

PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen

Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLe(x)) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe(x) antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with (3)H-monosaccharide precursors to detect low-abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLe(x) antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing the SLe(x) antigen.

Generation of Rat Monoclonal Antibodies Against Murine LAIR-1

The leukocyte-associated Ig-like receptor 1 (LAIR-1), an inhibitory receptor bearing two immunoreceptor tyrosine-based inhibitory motifs (ITIM), is expressed on the majority of peripheral leukocytes, including NK cells, T cells, B cells, monocytes, dendritic cells, granulocytes, and thymocytes and is involved in immunologic regulation and hematopoiesis. Murine LAIR-1 (mLAIR-1) is the homolog molecule of human LAIR-1. Using mLAIR-1-Fc as the immunogen and the technique of rat B lymphocyte hybridoma, we raised three hybridoma cell lines secreting monoclonal antibodies (MAb) to mLAIR-1, designated FMU-mLAIR-1.1, -1.2, and -1.3. Rat immunoglobulin class and subclass of the MAb FMU-mLAIR-1.1 approximately 3 were determined to be IgM, IgG1, and IgM, respectively. All these MAbs can bind the mLAIR-1 in immunocytochemistry and immunohistochemistry. FMU-m LAIR-1.2 worked well not only in Western blot assay but also in recognizing natural LAIR-1 molecules on the surface of P388D1, J774, and WEHI3 cells, and mLAIR-1 cDNA-transfected CHO cells detected by FCM. Thus, successful production of rat anti-murine LAIR-1 monoclonal antibodies provides a new powerful tool for investigation of murine LAIR-1 function in mouse model, both in vitro and in vivo.

Co-regulated decrease of Neurokinin-1 receptor and Hemokinin-1 gene expression in monocytes and macrophages after activation with pro-inflammatory cytokines

Hemokinin-1 (HK-1), a potent ligand for the Neurokinin-1 receptor (NK-1) is thought to play a role in the immune system. To investigate the regulation of this receptor–ligand pair, we examined the effects of pro-inflammatory cytokines on their expression in the monocyte/macrophage cell lines Wehi-3 and RAW264.7. We demonstrate co-expression of NK-1 and HK-1 mRNA in both lines, as well as functional NK-1 receptor protein in Wehi-3 cells. Stimulation with IFN-γ, IL-1β and TNF-α markedly decreased NK-1 and HK-1 mRNA as well as NK-1 receptor protein, which coincided with monocytic differentiation. A co-regulated decrease could also be observed in differentiating primary bone marrow macrophages, suggesting that this receptor–ligand pair may be controlled by cytokine networks and may serve a developmental role in the immune system.

Diallyl sulfide inhibits WEHI‐3 leukemia cells in Blab/C mice In Vivo

Diallyl sulfide (DAS), an organosulfur compound from garlic, is recognized as a potential chemo-preventive agent, and has a wide range of pharmacological effects, including anti-leukemia cells in vitro. Many investigators have already demonstrated that DAS induces apoptosis from human leukemia HL-60 and K562 leukemic cells, but there are no reports regarding whether or not it affects leukemia cells in vivo. However, the exact effect of DAS on leukemia cells in murine in vivo is unknown. In this study, we focused on the effects of DAS in vivo affecting murine leukemia WEHI-3 cells. The primary studies tested the effects of DAS on the changes of percentage of viable cells of WEHI-3 in vitro by MTT assay and the results showed that DAS decreased the % of viable cells and those effects are dose-and time-dependent manners. The following experiments examined the effects of DAS on leukemia WEHI-3 cells in BLAB/c mice in vivo after i.p. was injected with WEHI-3 cells before being cotreated with or without DAS, and the results indicate that DAS decreased the percentage of Mac-3 marker, inhibiting the differentiation of precursor of macrophage and T cells. The final experiments measured the weights of liver and spleen and the results indicated that DAS decreased the weights of liver and spleen. It also brought attention to the enlarged spleens in murine after i.p. was injected with WEHI-3 cells. However the DAS decreased the size of the spleen. The results showed that DAS affects WEHI-3 cells in vitro and in vivo. Further investigations are needed to find out the molecular mechanism of DAS on the leukemia mice in vivo.

Rutin inhibits WEHI‐3 leukemia cells in Balb/C mice in vivo

Rutin, one of the flavonoids, possesses a wide range of biological effects, including antitumor, antioxidant, and anti-inflammatory activities and anti-leukeima cells. Although rutin had been reported to induce apoptosis via caspase-3 activation on human leukemia K562 cells, there is no available information to address whether or not rutin affects leukemia cells in vivo. However, the exact effect of rutin on leukemia cells on murine in vivo is undefined. Therefore, in the present we are focused on the effects of rutin in vivo affecting WEHI-3 cells on murine leukemia. These experiments were divided into 3 parts. Part I, we examined the effects of rutin on the percentage of viable cells by flow cytometry on WEHI-3 cells in vitro and the results indicated that rutin decreased the viable cells and those effects are dose-and time-dependent effect manners. Part II, we examined the effects of rutin on leukemia WEHI-3 cells in BLAB/c mice in vivo and the results indicated that rutin decreased the percentage of Mac-3 marker, inhibiting the differentiation of precursor of macrophage and T cells which were isolated from spleen. Part III, we examined the size and weight changes of liver and spleen and the results demonstrated that rutin decreased the size and weight of liver and spleen when comparing to the control group. One of the characters of leukemia is the enlarged spleen in murine after i.p. was injected with WEHI-3 cells. Part IV, in the pathological examination, rutin affected the liver and spleen which came from the BLAB/c mice i.p. after injected with WEHI-3 cells treated with or without rutin. Our result showed that rutin affects WEHI-3 cells in vitro and in vivo.

Quercetin promoted natural killer cells activity and inhibits WEHI‐3 leukemia cells in Balb/C mice in vivo

Dietary polyphenols have been demonstrated to reduce riskof chronic diseases such as cancer, but the precise underlyingmechanism of protection remains unclear. Quercetin, one of the flavonoids found in onion, grapes, green vegetables, etc., possesses a wide range of biological effects including potent antiproliferative effects against various malignant cells. However, the exact effect of leukemia cells on murine in vivo is undefined. The aim of this studywas to investigate the effect of quercetin on affecting murine leukemia WEHI-3 cells in vivo. The cytotoxicity analysis demonstrated that quercetin inhibited the growth of WEHI-3 cells in dose- and time- dependent manners. After animals were injected with i.p by WEHI-3 cells that were oral treated with quercetin, the animals were sacrificed to measure the body weight and the results demonstrated that quercetin decreased both the size and weight of liver and spleen. Based on the ELISA analysis, it was demonstrated that quercetin decreased the percentage of Mac-3 marker and it also inhibited the differentiation of precursor of macrophage and T cells. Based on the pathological examination after animals were injected with i.p. through WEHI-3 with or without orally treated quercetin, it was demonstrated that quercetin did affect spleen and liver tissues. Therefore, the function of quercetin was observed in the liver and spleen of mice previously injected with WEHI-3 cells. The results from flow cytometric analysis also demonstrated that quercetin promoted the killing target cells of natural killer cells from mouse. Apparently, our results demonstrated that quercetin affects WEHI-3 cells in vitro and in vivo.

Wogonin inhibits WEHI‐3 leukemia cells in Blab/C mice In Vivo

Wogonin is one of the flavonoids and it has been shown to exert antioxidant, anti-viral, anti-bacteria, anti-thrombotic, anti-inflammatory and cytostatic activities. Recently, it also has been reported that wogonin induced apoptosis of human leukemia cells. Importantly, wogonin lacks toxicity for normal peripheral blood mononuclear cells. However, there is no available information to address whether or not wogonin affects leukemia cells in vivo. In this study, we investigated the effects of wogonin in vivo affecting murine leukemia WEHI-3 cells. This study is divided into in vitro and in vivo, the in vitro studies, it indicated that wogonin inhibit cell proliferation of WEHI-3 using MTT assay and those effects are dose-and time-dependent manners. The in vivo studies, the major assay is flow cytometry and the results indicated wogonin inhibit the percentage of Mac-3 marker, inhibiting the differentiation of precursor of macrophage and T cells; wogonin decrease the weights of liver and spleen after comparying to non-treated groups; wogonin decreased the sizes of liver and spleen. The results indicated that wogonin affects WEHI-3 cells in vitro and in vivo. Wogonin may be used for therapy of leukemia.

In vitro co-stimulation of anti-tumor activity by soluble B7 molecules.

In order to investigate the anti-tumor activity of a soluble B7-1/immunoglobulin G fusion protein and explore an effective method to eliminate immune escape of tumor cells, a recombinant vector encoding this fusion protein was constructed and constitutively expressed in Chinese hamster ovary cells. After purification with protein G affinity chromatography, the soluble fusion protein was tested for bioactivity. Results showed that the fusion protein could significantly increase the density of B7-1 molecules on WEHI-3 cells, a mouse leukemia cell line. Through allogeneic mixed lymphocyte tumor cultures, it was demonstrated that, with the presence of the first signal, it could also significantly enhance T cell activation and killing activity against WEHI-3 cells and interleukin-2 secretion by activated mouse T lymphocytes. The conclusion can be drawn that the soluble B7-IgG fusion protein has a potent capacity to generate or enhance anti-tumor immune response in vitro, and its clinical value deserves further investigation.

Initial study on immune escape mechanism of mouse acute myelomonocytic leukemic cell line WEHI-3

Objective: To investigate the expression of Fas, Fas ligand (FasL) and CD80 on the cell surface of mouse acute myelomonocytic leukemia cell line WEHI-3 and the function of FasL. Methods: The expression of Fas, FasL and CD80 was detected on WEHI-3 cell surface by flow cytometry. Simultaneously the function of FasL was determined by Thymidine (3H-TdR) Incorporation. Results: The expression of CD80 and Fas on WEHI-3 cell surface was 5.06%±0.41% and 6.75%±2.31% (n=5) respectively, and the expression of FasL was up to 63.73%±5.23% (n=5). The apoptotic rate of YAC-1 cells was 26%±4.5%, 35%±3.2% and 43%±2.7% (n=5) respectively when WEHI-3 (effector cell, E) and Fas+ YAC-1 cells (target cell, T) were cultured in the ratio of 3:1, 10:1 and 30:1. Conclusion: WEHI-3 cells express high FasL, low Fas and CD80, and can induce apoptosis of Fas+ YAC-1 cells.

Glypican-1 Is Frequently Overexpressed in Human Gliomas and Enhances FGF-2 Signaling in Glioma Cells

Signaling by fibroblast growth factor 2 (FGF-2), an autocrine stimulator of glioma growth, is regulated by heparan sulfate proteoglycans (HSPGs) via a ternary complex with FGF-2 and the FGF receptor (FGFR). To characterize glioma growth signaling, we examined whether altered HSPGs contribute to loss of growth control in gliomas. In a screen of five human glioma cell lines, U118 and U251 cell HSPGs activated FGF-2 signaling via FGFR1c. The direct comparison of U251 glioma cells with normal astrocyte HSPGs demonstrated that the glioma HSPGs had a significantly elevated ability to promote FGF-2-dependent mitogenic signaling via FGFR1c. This enhanced activity correlated with a higher level of overall sulfation, specifically the abundance of 2S- and 6S-containing disaccharides. Glioma cell expression of the cell-surface HSPG glypican-1 closely mirrored the FGF-2 coactivator activity. Furthermore, forced expression of glypican-1 in (glypican-1-deficient) U87 glioma cells enhanced their FGF-2 response. Immunohistochemical analysis revealed a highly significant overexpression of glypican-1 in human astrocytoma and oligodendroglioma samples compared with nonneoplastic gliosis. In summary, these observations suggest that altered HSPGs contribute to enhanced signaling of FGF-2 via FGFR1c in gliomas with glypican-1 playing a significant role in this mitogenic pathway.

Berberine Inhibits WEHI‐3 Leukemia Cells in Balb/C Mice In Vivo

Berberine, an isoquinoline alkaloid, has a wide range of pharmacological effects, including anti-leukeima cells. Yet the exact effect on murine in vivo is unknown. Although berberine had been reported to induce apoptosis on human leukemia HL-60 and murine WEHI-3 cells, there are no reports regarding whether or not it affects leukemia cells in vivo. Therefore, the present studies are focused on the effects of berberine in vivo affecting murine leukemia WEHI-3 cells. At first, we re-examined the effects of berberine on the percentage of viable cells on WEHI-3 cells in vitro and the results showed that berberine induced dose-and time-dependent effect manners. Second, we then examined the effects of berberine on leukemia WEHI-3 cells in BLAB/c mice and the results indicate that berberine decreased the percentage of Mac-3 marker, inhibiting the differentiation of precursor of macrophage and T cells. Third, we examined the weight of liver and spleen and the results indicated that berberine decreased the weight of liver and spleen. However, the important character of WEHI-3 leukemia is the enlargely spleens in murine after i.p. injected WEHI-3 cells. Fourth, based on the pathological examination we did see the function of berberine on the liver and spleen which came from the BLAB/c mice i.p. after injected by WEHI-3 cells. Our result showed that berberine affects WEHI-3 cells in vitro and in vivo. Further investigations are needed to find out the molecular mechanism.

Down‐regulation of cyclin B1 expression by berberine promotes leukemia cells entry into G2/M phase of cell cycle

Berberine has a wide range of biological actions that suggest it may be of use in cancer prevention. It was reported previously that berberine induced cell cycle arrest not only at the G0/G1 phase but also at the G2/M phase dependent on the dose. However, the mechanism of berberine-induced G2/M phase arrest in leukemia has not been fully understood. In the present, the effects of the naturally occurring berberine (the major constituent of Coptis Chinese) on growth, cell cycle, and cyclins A, E, and B1 expression were investigated in the human promyelocytic leukemia HL-60 cells and murine myelomonocytic leukemia WEHI-3 cells. The flow cytometry assays indicated that berberine induced cytotoxicity and G2/M arrest in both examined cell lines. Berberine induced G2/M arrest in both examined cell lines were accompanied by increased levels of Wee1 and decreased the Cdc25c, Cdk1 and cyclin B1, but it did not affect CDK2 which were examined by Western blotting assay. Therefore, berberine induced G2/M arrest in both examined cell lines through the inhibition of activation of cyclin B1.

Peptidomics: A modern approach to biodiversity of peptides

Abstract Murine myelomonocytes WEHI-3 and human erythroid leukemia K562 cells were shown to release peptides into the surrounding medium. Four N-terminal sequences of the peptides derived from unknown protein precursors were identified in the conditioned medium of WEHI-3 culture. Twelve N-terminal sequences of the peptides released by K562 cells were established. K562 cells were shown to release long hemoglobin α- and ε-chains fragments, as well as peptides derived from carbonic anhydrase XI, melanophilin, cadherin EGF LAG G-type receptor 2, aldolase A, melastatin 1, peptide homologous to ryanodine receptor fragment, and a few peptides derived from the proteins with unknown function. Comparative analysis of the peptides released by the intact erythroid leukemia K562 cells and the same cells after differentiation (induced by guanosine monophosphate, GMP, and accompanied by start of hemoglobin biosynthesis), has demonstrated that the level of six components, including all established hemoglobin fragments, carbonic anhydrase fragment, and the fragment of the unknown protein significantly increased in the course of differentiation. The spectrum of the peptides released by K562 cells strongly differed from that of the mature erythrocytes: (1) in contrast to the mature erythrocytes, K562 cells release long α-globin fragments and the fragment of ε-globin; (2) erythrocytes almost exclusively release the products of hemoglobin proteolysis; in the case of K562 cells the fragments of enzymes, receptors, and other functional proteins were found. So, the concentration and the content of the individual peptides released by the cells depend on the differentiation state. The comparative analysis of the dynamics of peptide secretion by human erythrocytes performed in the presence and the absence of the 3 % glucose has shown that the rate of the peptide release strongly depends on cell metabolism. In summary, peptidomic studies of cell cultures provide valuable information on the mechanisms of peptide pool formation in tissues and the whole organism.

Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-κB in A2A adenosine receptor induction

The A(2A) adenosine receptor (A(2A)AR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A(2A) agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism initiated through NF-kappaB to terminate the activation of human and mouse macrophages.

Comparative analysis of proapoptotic activity of cytochrome c mutants in living cells

A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and "yeast --> horse" hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 +/- 0.5 microM (47 +/- 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The "yeast --> horse" hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis.

Experimental Study on Apoptosis in Leukemia Cells Induced by Econazole

To investigate apoptosis of mouse leukemia cell (WEHI-3) induced by econazole and its mechanism, apoptosis induced by econazole was examined by flow cytometry, while free calcium ([Ca(2+)]i) was determined by Fura-2 fluorescein load technique. The protein was isolated from endoplasmic reticulum of WEHI-3 cells, and then the expression of caspase-12 and caspase-7 was evaluated by Western blot. The results showed that WEHI-3 exhibited typical change of apoptosis when it was treated by econazole, [Ca(2+)]i was significantly higher in comparison with the control. The expression of caspase-12 and caspase-7 enhanced as the econazole concentration increased. In conclusion, econazole can induce WEHI-3 cell apoptosis and the caspase-12 plays a key role in this process.

Glycan Array Screening Reveals a Candidate Ligand for Siglec-8*

Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is selectively expressed on human eosinophils, basophils, and mast cells, where it regulates their function and survival. Previous studies demonstrated sialic acid-dependent binding of Siglec-8 but failed to reveal significant substructure specificity or high affinity of that binding. To test a broader range of potential ligands, a Siglec-8-Ig chimeric protein was tested for binding to 172 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. Of these, approximately 40 structures were sialylated. Among these, avid binding was detected to a single defined glycan, NeuAcalpha2-3(6-O-sulfo)Galbeta1-4[Fucalpha1-3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex. Notably, neither unsulfated sLex (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLex, NeuAcalpha2-3Galbeta1-4[Fucalpha1-3](6-O-sulfo)GlcNAc) supported detectable binding. Subsequent secondary screening was performed using surface plasmon resonance. Biotin glycosides immobilized on streptavidin biosensor chips were exposed to Siglec-8-Ig in solution. Whereas surfaces derivatized with sLex and 6-sulfo-sLex failed to support detectable Siglec-8 binding, 6'-sulfo-sLex supported significant binding with a Kd of 2.3 microm. In a separate test of binding specificity, aminopropyl glycosides were covalently immobilized at different concentrations on activated (N-hydroxysuccinimidyl) glass surfaces (Schott-Nexterion Slide H). Subsequent exposure to Siglec-8-Ig precomplexed with fluorescein isothiocyanate anti-human Fc resulted in fluorescent signals at immobilized concentrations of 6'-sulfo-sLex of <5 pmol/spot. In contrast, sLex and 6-sulfo-sLex did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot). We conclude that Siglec-8 binds preferentially to the sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl.

IL-3 derived from CD4+ T cells is essential for the in vitro expansion of mast cells from the normal adult mouse spleen.

A null cell line (SCM1) was established by a culture of spleen cells (SC) from normal adult C57B1/6 mice with complete medium alone for 10 days and followed by weekly cultures with a 25% WEHI-3 cell culture supernatant. Phenotype analysis showed that the SCM1 cells were negative for CD3, Thy1.2, B220, Mac-1, Gr-1, NK1.1 and MHC class II, but were positive for MHC class I, Fc gamma RII/ III, Fc epsilon RI, c-kit and the receptor against wheat germ agglutinin. These findings suggested that the SCM1 cells were mast cells. In an in vitro proliferation assay. SCM1 cells proliferated in the presence of either IL-3 or stem cell factor (SCF), but not in the presence of IL-4, whereas IL-4 showed an augmenting effect on their proliferation in the presence of either IL-3 or SCF. In analysing the mechanism by which such mast cells could be expanded from normal adult mouse SC, the addition of anti-IL-3 MoAb, but not anti-SCF MoAb, into the initial culture inhibited the subsequent expansion of either IL-3-or SCF-responding cells. The prior depletion of CD4+ T cells abrogated the capacity of the SC to enhance the expansion of SCF-responding cells, and this inability was restored by the addition of IL-3. Moreover, the culture supernatant of normal adult SC alone contained considerable levels of IL-3. Taken together, our findings suggest that, in an in vitro culture, CD4+ T cell-derived IL-3 therefore enhances the expansion of mast cells from the normal adult mouse spleen.

Discordant expression of selectin ligands and sialyl Lewis x–related epitopes on murine myeloid cells

Murine leukocytes are thought to express alpha2-3-sialylated and alpha1-3-fucosylated selectin ligands such as sialyl Lewis x (sLe(x)), although monoclonal antibodies (mAbs) to sLe(x) or Le(x) reportedly do not bind to murine leukocytes. We observed that P- and E-selectin bound to pronase-sensitive ligands on murine monocytic WEHI-3 cells and murine neutrophils, indicating that the ligands for both selectins are glycoproteins. CSLEX-1, HECA-452, and other widely used mAbs to sLe(x) and Le(x) did not bind to WEHI-3 cells and bound at very low levels to murine neutrophils. Only the anti-sLe(x) mAbs 2H5 and KM93, which also recognize nonfucosylated glycans, bound to WEHI-3 cells. 2H5 and KM93 bound to pronase-resistant structures, indicating that the mAbs did not identify selectin ligands. Treatment of WEHI-3 cells with glycosidases or chlorate demonstrated that sialic acid modifications, alpha1-3-galactosylation, or sulfation did not mask epitopes for mAbs to sLe(x) or Le(x). Compared to human promyelocytic HL-60 cells, WEHI-3 cells and murine neutrophils expressed low alpha1-3-fucosyltransferase activities. Consistent with very low endogenous fucosylation, forced fucosylation of intact WEHI-3 cells or murine neutrophils by exogenous alpha1-3-fucosyltransferase FTVI and GDP-fucose created many new epitopes for anti-sLe(x) mAbs such as HECA-452 and CSLEX-1. Nevertheless, forced fucosylation of intact cells did not significantly augment their ability to bind to fluid-phase P- or E-selectin or to roll on immobilized P- or E-selectin under flow. These data suggest that murine myeloid leukocytes fucosylate only a few specific glycans, which interact preferentially with P- and E-selectin.