Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Purification of immunoglobulin production stimulation factor II α derived from Namalwa cells

An immunoglobulin production stimulating factor (IPSF) in human lymphoblastoid Namalwa cells was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction chromatography and gel filtration, and named IPSF-II . IPSF-II was estimated as a 112 KD protein composed of a 40 KD polypeptide and two 36 KD polypeptides. The 36 KD protein extracted from SDS-polyacrylamide gel showed IPSF activity, but not the 40 KD protein. The IPSF activity was reasonably stable in alkaline but unstable in acidic solution and heat-unstable. In a serum-free medium, IPSF-II stimulated IgM production of human-human and mouse-mouse hybridomas 4–15 and 2-fold, respectively, although its growth stimulatory effect on hybridomas was negligible. The factor did not stimulate IgG production in either human or mouse hybridomas in the same serum-free medium. These results suggested that IPSF-II was a new cellular factor for stimulating IgM productivity of hybridomas.Abbreviations Ig immunoglobulin - MAb monoclonal antibody     

Immunoglobulin production stimulating and inhibiting factors derived from human lung adenocarcinoma PC-8 cells

Addition of a 3 M KCl extract of a human lung adenocarcinoma PC-8 cells to the culture media of lymphocytes, which were isolated from normal donors and from lung or breast cancer patients, elevated immunoglobulin (Ig) production by 3–5 times in the presence of pokeweed mitogen. However, addition of higher concentration of the extract inhibited Ig production and proliferation of lymphocytes. The Ig production stimulating factor (IPSF) was separated from the inhibiting factor, using 50% ammonium sulfate precipitation. Upon chromatofocusing, IPSF activity was detected mainly in the pH 4.5 fraction but minor activity was also detected in other pH fractions. IPSF also enhanced Ig production of a B-lymphoblastoid cell line transformed by Epstein-Barr virus and a human-human hybridoma, by more than 2-fold. This suggests that IPSF interacts with B-lymphocytes directly to enhance their Ig production. IPSF activity was also detected diversely in human lung squamous carcinoma QG56, human B-lymphoblastoid HO-323, and a T cell line CEM.     

Increase of immunoglobulin productivity of human-human hybridoma HB4C5 cells by histone

A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt's lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin production-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much. These facts strongly suggest that histone acts as an immunoglobulin production-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.     

Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes

Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 μg/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 °C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor. GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ig - immunoglobulin; IPSF - immunoglobulin production stimulating factor; PBL - peripheral blood lymphocytes; HPLC - high-performance liquid chromatography. This revised version was published online in July 2006 with corrections to the Cover Date.     

Screening of Immunoglobulin Production Stimulating Factor (IPSF) in Foodstuffs Using Human-human Hybridoma HB4C5 Cells

We screened the immunoglobulin production stimulating factor (IPSF) in foodstuffs, using human-human hybridoma HB4C5 cells cultured in serum-free 1TES-ERDF medium, and found that egg yolk lipoprotein (YLP), lactoferrin, Block Ace, and casein had IPSF activity. The maximum IPSF activity was obtained at concentrations over 100^g/ml in YLP, 10μg/ml in lactoferrin, and 25 μg/ml in Block Ace and casein. These IPSFs stimulated the IgM production of human-human and mouse-mouse hybridomas, but their effect on IgG producers was very small. This suggests that IgG production of hybridomas is regulated differently from their IgM production.     

Heat denaturation enhanced immunoglobulin production stimulating activity of lysozyme from hen egg white

Lysozyme from hen egg white was identified as an immunoglobulin production stimulating factor (IPSF) that enhances immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of lysozyme was facilitated by heat treatment. The heat treatment of lysozyme at 83 degrees C for 30 min activated its specific IPSF effect 30.0-fold compared with that of native lysozyme. The IPSF activity of lysozyme heat-treated at 83 degrees C in 4 M urea solution was enhanced 8.4-fold than that of native lysozyme. However, lysozyme that was not heated in 4 M urea solution completely lost its IPSF activity. This means that the IPSF activity of this enzyme in 4 M urea was reactivated by thermal treatment. Moreover, coexistence of 0.5 mM 2-mercaptoethanol (2-ME) during heating in 4 M urea solution extremely enhanced the IPSF activity up to 77.8-fold. The uptake of lysozyme by hybridoma cells was enhanced by heat denaturation in 4 M urea. The hydrophobicity of lysozyme was extremely increased by heat-treatment in 2-ME containing urea solution. It is expected from these findings that the increase in the hydrophobicity caused the enhancement of incorporation of lysozyme into target cells, and resulted in the acceleration of IgM production.     

Stimulation of IgM production in human-human hybridoma HB4C5 cells by chitosan.

We screened for immunoglobulin production stimulating factors (IPSFs) in polysaccharides using human-human hybridoma cells, HB4C5, cultured in serum-free medium. Among polysaccharides, citrus pectin, locust bean gum, and chitosan stimulated IgM production of HB4C5 cells. Especially chitosan showed the strongest IPSF activity; 100 ng/ml of chitosan stimulated IgM production approximately 5-fold. Chitosan had several characteristics as IPSF, as follows. 1) For the IPSF activity, 70-90% deacetylation was essential. 2) Chitosan oligomers (n = 5, 6, 7) and chitin oligomers (n = 5, 6, 7) showed no IPSF activities. 3) The IPSF activity of chitosan was inhibited by glucosamine, one of the constitutive sugars of chitosan. 4) Chitosan stimulated IgM production of human lymphocytes in serum-free culture, but not IgG or IgA, nor in serum-supplemented culture.     

Monoclonal antibody specific for T cell-derived human IgE binding factors

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.     

Purification and characterization of immunoglobulin production stimulating factor-IIβ derived from Namalwa cells

Two immunoglobulin production stimulating factors (IPSF) have been found in human Burkitt's lymphoma Namalwa cells. One IPSF named IPSF-II was purified and identified as glyceraldehyde-3-phosphate dehydrogenase as previously reported. We report here purification, identification and characterization of IPSF-II. IPSF-II was purified by the serial use of ammonium sulfate fractionation, hydrophobic interaction column chromatography, anion-exchange column chromatography and gel filtration. The IPSF-II was estimated as a 46 KD monomeric polypeptide by gel filtration and SDS-PAGE. Partial amino acid sequence of the 46 KD protein was analyzed for 26 amino acid residues. The sequence very closely coincided with enolase (EC 4.2.1.11) derived from various origins and, it was completely homologous with that of human enolase -chain. Rabbit muscle enolase stimulated IgM production of hybridoma lines, and IPSF-II had the enzymic activity. These results suggested that IPSF-II was -enolase or its isozyme. IPSF activities of IPSF-II was stable in alkaline conditions whereas the enzymic activity was rapidly lost in alkaline conditions. Though IPSF-II stimulated IgM production of both human-human and mouse-mouse hybridoma lines in serum-free condition, it partially suppressed IgE production of mouse-mouse hybridoma lines.     

Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 μg/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400–7000 bp slightly stimulated IPSF activity at 0.06 μg/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell. This revised version was published online in August 2006 with corrections to the Cover Date.     

Recombinant perchloric acid-soluble protein suppresses the immunoglobulin production of human-human hybridoma HB4C5 cells

Because perchloric acid-soluble protein (PSP) has been conserved evolutionally in various species from Escherichia coli to humans, it may reflect an involvement in basic cellular regulation. However, the precise function of PSP is currently unknown. In this study, we examined the direct effect of PSP on the production of immunoglobulin (Ig) using human B, HB4C5, NAT-30, and U266 cells because it has been reported that subcutaneous administration of PSP affects rodent immune systems. Suppression of Ig productivity and decrement of the cell viability was recognized only in HB4C5 cells by the addition of PSP into the medium. On the other hand, PSP had no effect on Ig productivity and cell viability in NAT-30 and U266 cells. In addition, PSP was clearly incorporated by HB4C5 but not by the other cells. These results suggest that the Ig production suppressed by PSP, which has been previously reported to inhibit protein synthesis, contributed to the incorporation of PSP into the HB4C5 cells.     

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 g/ml lactoferrin, 10M ethanolamine, 35 /ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.Abbreviations MoAb monoclonal antibody     

Burkitt lymphoma cell lines are prone to recombination in the switch region of the Ig mu heavy chain locus

Different recombinations have been found at the Ig heavy chain gene loci in a number of sublines of the Burkitt lymphoma (BL) cell line Namalwa, following prolonged in vitro culture. The Namalwa sublines examined are DNA fingerprint-identical and derived from a monoclonal source. Recombinant DNA clones were used to map the Ig heavy chain gene mutations to a region between the VDJ and C mu segment of the locus. This region is associated with Ig heavy chain class switching in normal B cells. Of 24 clones established from one subline, three were found to have additional VDJ-C mu region mutations, indicating a high frequency of mutation at this locus.     

The influence of mesenchymal stromal cells on B-cell line growth and immunoglobulin synthesis

There are conflicting data on the influence of mesenchymal stromal cells (MSCs) on B-lymphocyte growth, differentiation, and immunoglobulin (Ig) production. The aim of this study was to investigate the influence of MSCs derived from the adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of the lymphoblastoid cell line Namalva and myeloma cell line U266. Cocultivation of Namalva cells with MSCs stimulated their proliferation and decreased the doubling time and minimal inoculation dose necessary for cloning of these lymphoblastoid cells. The presence of MSCs supported the survival and proliferation of Namalva cells cultivated in growth factor-deficient medium. MSCs also stimulated the proliferation of U266 myeloma cells. MSCs derived from adipose tissue of both healthy donors and patients with breast cancer effectively stimulated cell proliferation of B-cell lines. The presence of MSCs in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells, respectively. Cocultivation of Namalva or U266 cells with MSCs provided tight contacts between cells of both types.     

Production of human suppressor T cell hybridomas

To study human T cell suppression of immunoglobulin (Ig) synthesis with homogeneous populations of immunoregulatory cells, human suppressor T cell hybridomas were prepared by somatic cell fusion of concanavalin A-activated peripheral blood T cells with hypoxanthine-guanine phosphoribosyltransferase-(HGPRT, EC 2.4.2.8) deficient human leukemic CEM T cells. After selection in hypoxanthine-aminopterin-thymidine (HAT) medium and cloning by limiting cell dilution, two human T cell hybridomas were identified that produced 60 to 80% suppression of in vitro polyclonal immunoglobulin production when cocultured with pokeweed mitogen- (PWM) stimulated peripheral blood lymphocytes. Further, one of the suppressor T cell hybridomas constitutively secreted a soluble suppressor factor(s) (TsF) of m.w. 70,000 to 85,000 daltons, which produced reversible noncytotoxic inhibition of lectin-activated B cell Ig production. In contrast, this TsF did not inhibit lectin- or antigen-induced T cell proliferation, nor did it interfere with the generation or effector function of cytotoxic T cells. Additional studies indicated that this Tsf acts directly on B cells or monocytes rather than indirectly modulating the activity of immunoregulatory T cells. In summary, these studies suggest that techniques of somatic cell fusion may provide a valuable approach to further study human immunoregulatory cell-cell interactions as well as provide a source of sufficient quantities of important lymphokines for further purification and characterization.     

Human peripheral blood monocytes release a 30,000 dalton factor (30 KD MF) that stimulates immunoglobulin production by activated B cells

Supernatants (SN) of well-washed adherent human monocytes, obtained from T cell-depleted peripheral blood mononuclear cells, contain a 30,000 dalton protein (30 KD MF) that increases immunoglobulin (Ig) synthesis by EBV-activated B cells two- to fourfold. This factor is released spontaneously during the first 20 hr after monocytes are placed in culture. SN containing 30 KD MF are inactive in the thymocyte co-stimulator assay, under conditions that will detect as little as 0.5 U of purified IL 1. The addition of autologous T cells to isolated adherent monocytes, previously depleted of T cells, suppresses the release or activity of this B cell stimulator in a dose-dependent manner. In addition, 30 KD MF stimulates a two- to fourfold increase in IgA production by cells of an EBV-transformed B cell line (JB/FF line) without increasing incorporation of [3H]thymidine. In contrast, stimulation of this B cell line with up to 10 U of purified IL 1 increases IgA synthesis by less than 50%, and addition of up to 100 U of recombinant IL 2 causes no change whatsoever in IgA production. However, co-stimulation with 30 KD MF and recombinant IL 2 or recombinant gamma-interferon induces more Ig production than is caused by the monocyte factor alone. These observations suggest that the monocyte, in addition to acting as an antigen-presenting cell and source of IL 1, facilitates B cell differentiation by producing a factor which acts both independently and in synergy with cytokines produced by T cells to stimulate Ig production by B lymphocytes.     

The Ba fragment of complement factor B inhibits human B lymphocyte proliferation

Normal human B lymphocyte function is finely regulated by both positive and negative signals at each stage of activation, proliferation, and differentiation. Activation signals include antigen and surface Ig cross-linking agents such as anti-mu or anti-delta. Signals inducing proliferation include IL-2, high m.w.-B cell growth factor (BCGF), and low m.w.-BCGF. IL-2 as well as IL-6 and other partially characterized B cell differentiation factors can induce terminal differentiation of proliferating B cells into Ig-secreting plasma cells. Various C components have been described to regulate B cell function including Bb that enhances proliferation, C5a that enhances Ig production, and C3a that inhibits Ig production. In our study, we examined the ability of the factor B cleavage fragment Ba to influence human B cell function. Ba did not affect the activation of resting B cells but inhibited the proliferation of activated B cells stimulated with either high m.w.-BCGF or low m.w.-BCGF. The inhibition occurred with doses of Ba as low as 1 microgram/ml (29 nM). Ba was found to bind to activated human B lymphocytes in a saturable manner with an apparent K of approximately 25 nM and an apparent Bmax of 56,000 sites/cell. A peptide made of the carboxy terminal 10 amino acids of Ba (GHGPGEQQKR), was also found to inhibit growth factor induced proliferation of activated B cells but at an ID50 of approximately 5 microM. Finally, Ba was found to inhibit the terminal differentiation of Staphylococcus aweus Cowan-activated B cells stimulated with B cell differentiation factors but not Ig secretion by the partially differentiated EBV-transformed cell line SKW.6. Thus, concentrations of Ba achievable in vivo at sites of active inflammation were found to act on human B lymphocytes by inhibiting their proliferation. This may act to limit the immune response to a specific antigenic challenge.