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.     

Modification of cord blood IL-6 production with IgM enriched human immunoglobulin in term and preterm infants

Pro-inflammatory cytokines contribute significantly to the morbidity of premature infants. IL-6 and IL-8 are involved in the pathogenesis of pulmonary and cerebral tissue injury. The effect of human immunoglobulin preparations on cytokine production in preterm infants has not been studied. We investigated the influence of immunoglobulin on LPS stimulated IL-6 and IL-8 production in cord blood of healthy preterm neonates. Ten non-infected preterm infants delivered by cesarean section and 5 healthy term neonates were included. In the preterm infants, significant IL-6 production was observed in the absence of immunoglobulin after 4 h [median 113 (39-725) pg/ml], 8 h [375 (234-1795) pg/ml] and 12 h [360 (248-2765) pg/ml] of LPS incubation. IL-6 concentrations were significantly lower after incubation with LPS+immunoglobulin after 4 h [median 38 (5-568) pg/ml; p=0.005], 8 h [178 (10-1830) pg/ml; p=0.001] and 12 h [182 (29-2530) pg/ml; p=0.002]. Cultures from term infants produced IL-6 levels approx. 4 times of those from premature infants unaffected by immunoglobulin. IL-8 production also correlated to gestational age and was not affected by immunoglobulin in both groups. Human immunoglobulin preparation may modify IL-6 production in cord blood cultures from premature infants.     

The regulatory roles of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production

A new mitogenic system for in vitro immunoglobulin production induced by tetanus toxoid is presented and the role of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production is investigated. Purified T, T4, T8, and B cells from normal individuals previously immunized but not boosted with tetanus toxoid were cultured in helper and suppressor assays and the number of immunoglobulin-secreting cells were enumerated after culture using a hemolytic plaque assay. The regulatory roles of T4 and T8 cells in this tetanus toxoid system were compared with the role of these subsets after pokeweed mitogen stimulation. Although most of the immunoglobulin produced in the tetanus toxoid system was polyclonal, there were differences in the time course, the magnitude of the responses, the radiosensitivity of the subsets, and optimal T- to B-cell ratios for immunoglobulin production which distinguish the tetanus toxoid and pokeweed mitogen systems.     

Jacalin, an IgA-binding lectin, inhibits differentiation of human B cells by both a direct effect and by activating T-suppressor cells

Jacalin, a lectin extracted from the seeds of Artocarpus intergifolia (jackfruit), has been reported to bind specifically to IgA while inducing B-cell polyclonal immunoglobulin secretion. We confirmed that jacalin only binds to IgA and not to IgG or IgM and extended these findings by showing that it does not bind to IgE. Addition of jacalin to either unfractioned peripheral blood lymphocytes or purified B cells failed to induce immunoglobulin synthesis; indeed immunoglobulin production was diminished in the presence of jacalin. We found that jacalin directly inhibited the induction of immunoglobulin synthesis from B cells in the presence of T-cell replacing factor. Cell lines making IgG, IgM, and IgA were inhibited by jacalin. Furthermore, T cells incubated with jacalin also inhibited immunoglobulin production by stimulated B cells. Under these conditions jacalin was found to be a potent mitogen for T cells but to induce little or no activation of B cells. Jacalin appears to be a potent T-cell mitogen which can induce suppressor T cells for Ig production. It also has a direct inhibitory effect on B-cell Ig production.     

Spermine enhances IgM productivity of human-human hybridoma HB4C5 cells and human peripheral blood lymphocytes

The polyamine spermine was assessed for enhancement of IgM production by human-human hybridoma, HB4C5 cells, under serum-free conditions. IgM production of HB4C5 cells was stimulated approximately 6-fold by the addition of 7.3 mM of spermine. The facilitating effect was observed soon after inoculation. In spite of suppression of cell growth, the IgM production rate was accelerated for at least 5 days without medium change. Moreover, laser confocal microscopic analysis revealed that the IgM content inside HB4C5 cells was increased by spermine treatment. These findings suggest that spermine enhances specific IgM productivity of the hybridoma line. Spermine also facilitated IgM production by human peripheral blood lymphocytes under serum-free conditions. This result implies that spermine enhances immunoglobulin production of not only specific hybridoma lines, but also non-specific immunoglobulin producers. Immunoglobulin production stimulating activity of spermine was accelerated 2-fold by the addition of DNA whith a chain length of about 400–7000 base pairs (bp). However, degraded short-chain DNA fragments (less than 200 bp) did not facilitate the immunoglobulin production stimulating activity of spermine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Immunoglobulin secreting cells in lymphocytes of patients with IgA deficiency or hyper-IgA

To assess the ability of immunoglobulin production in vivo, we enumerated the immunoglobulin secreting cells in the peripheral blood of patients with an IgA deficiency and of those with hyper-IgAemia. All seven patients with primary IgA deficiency and two of the three patients with secondary IgA deficiency had low numbers of IgA secreting cells. In all five patients with hyper-IgA the number of IgA secreting cells was increased. Our results suggest that measurement of immunoglobulin secreting cells in PBMCs is useful in the assessment of ability of immunoglobulin production in vivo.Abbreviations PBMCs peripheral blood mononuclear cells - MEM minimum essential medium - PFC plaque forming cells - VH immunoglobulin heavy chain variable region - CH immunoglobulin heavy chain constant region     

Methods of enhancing the radioprotective efficacy of serum immunoglobulin

In this work the authors propose three ways of increasing the radioprotective efficiency of immunoglobulin: in vitro irradiation with gamma-quanta (60Co, 20,000 Gy), increasing the time of storage of whole blood (raw material for immunoglobulin production) up to 5-7 days at +4 degrees C, and the application of immunoglobulin with antibiotics. In experiments in vitro it was first shown that a combination of immunoglobulin with gentamicin exerted a pronounced bactericidal effect on E. coli. The effect was absent with immunoglobulin and markedly less pronounced with gentamicin used separately.     

Differential effect of opioids on immunoglobulin production by lymphocytes isolated from Peyer's patches and spleen

The mucosal immune system plays an important role in blocking the penetration of invasive organisms into various mucosal surfaces. Evidence now suggests neuroendocrine peptide hormones have immunomodulatory properties, including the ability to alter mucosal immunity. The potential for opioid compounds and corticotropic hormone (ACTH) to modulate mucosal immune function was investigated. We have found beta-endorphin, ACTH, and naltrindole (delta-class opioid receptor antagonist) to significantly suppress concanavalin A-stimulated Peyer's patch lymphocyte immunoglobulin production of IgA, IgG, and IgM isotypes. Oxymorphindole, a delta class opioid receptor agonist, significantly decreased IgM but not IgA or IgG production by the mitogen-stimulated Peyer's patch lymphocytes. Both oxymorphindole and naltrindole modestly reduced interleukin-2 receptor expression of concanavalin A- (Con A)-stimulated splenic and Peyer's patch lymphocytes. Neither compound appreciably affected immunoglobulin production by lipopolysaccharide-stimulated Peyer's patch lymphocytes. Collectively, these results indicate stress-related peptides such as ACTH and opioids may be involved in the regulation of immunoglobulin synthesis by Peyer's patch lymphocytes.     

Pooled human immunoglobulin inhibits IL-4 but not IFN-gamma or TNF-alpha secretion following in vitro stimulation of mononuclear cells with Staphylococcal superantigen.

Intravenous immunoglobulin preparations have been successfully used in many disorders, where immunomodulation rather than immunoglobulin replacement has been the goal of therapy. The exact mechanisms by which immunoglobulin exerts its immunomodulatory effects are unclear. Proposed mechanisms include modification of T cell activation and alteration to cytokine production. As intravenous immunoglobulin therapy has been used in a number of disorders where superantigens are proposed to play a role in the disease pathogenesis, we have examined the effect of in vitro human pooled immunoglobulin on cytokine production from peripheral blood mononuclear cells in response to activation with the Staphylococcal superantigen Staphylococcal enterotoxin B. The authors found inhibition of secretion of interleukin 4 (IL-4) (P<0.001) but not interferon gamma (IFN-gamma) (P=0.13) or tumour necrosis factor alpha (TNF-alpha) (P=0.66) by pooled immunoglobulin at concentrations (6 g/l) which approximate the rise in serum immunoglobulin following in vivo IVIG therapy. Mononuclear cell proliferation was also inhibited by addition of pooled immunoglobulin to superantigen stimulated cultures. These effects do not relate to specific anti-staphylococcal enterotoxin B antibodies in the immunoglobulin preparation. The authors show that pooled human immunoglobulin can differentially modulate the secretion of IL-4 and IFN-gamma in response to superantigen stimulation.     

Proliferation and immunoglobulin secretion of lymphoblastoid cell lines are differently affected by soluble cytokines

Abstract. The aim of our study was to investigate whether supernatant from lipopoly-saccharide-activated monocytes (monocyte-factor) and/or cytokines could enhance secretion of human monoclonal antibodies specific to HLA antigens produced by Epstein—Barr virus lymphoblastoid cell lines (EBV-LCLs). In a low cell density culture system, the monocyte-factor significantly stimulated cell growth of three monoclonal and two polyclonal EBV-LCLs while no enhancement of immunoglobulin production was observed. The enhancement of proliferation was completely neutralized by an antiserum to human IL-6 suggesting that IL-6 was required for the stimulation of growth of LCLs. The effect of cytokines on proliferation showed large variations among the cell lines, with IL-l β generally inducing the highest response. Of the cytokines tested, only IL-2 was able to enhance total immunoglobulin secretion due to the induction of a higher production of light chains. The specific anti-HLA activity was slightly increased by IL-10 although this cytokine had no effect on total immunoglobulin concentration or proliferation.     

Blastogenesis and polyclonal immunoglobulin synthesis in murine spleen cells stimulated with lipopolysaccharide, lipid A and acid-degraded polysaccharide from Fusobacterium nucleatum

Lipopolysaccharide of Fusobacterium nucleatum strain Fevl was split by acid hydrolysis. The split products, i.e. lipid A and degraded polysaccharide were mitogenic for murine spleen cells as measured by uptake of [3H]thymidine. The uptake of [3H]thymidine was dose-dependent. Incubation of spleen cells with stimulants for 3 days resulted in a polyclonal activation of immunoglobulin synthesis. Higher mitogenic response and immunoglobulin production were found in spleen cells of athymic mice compared to those of thymic mice. The activity of lipid A in stimulating immunoglobulin synthesis was comparable with the parent lipopolysaccharide-Fevl, the degraded polysaccharide being the less potent stimulator.     

Cloning of mouse myeloma cells and detection of rare variants

Cultured mouse myeloma cells have been cloned in soft agar using a modification of the method established by Pluznik and Sachs ('65, '66) and by Bradley and Metcalf ('66). A linear relationship existed between the number of cells plated and the number of colonies produced. Conditions for obtaining optimum cloning efficiency and colony size were determined for the MPC-11 cell line. Feeder cells of mouse, human and rabbit origin and conditioned growth medium obtained from mouse cultures and had an enhancing effect on colony formation. Immunoglobulin production by cloned cells was detected by overlaying the clones with anti-immunoglobulin antiserum. The antiserum had no adverse effect on cloning efficiency or colony size. A reconstruction experiment was performed to show that the plate assay could reliably detect rare variants of immunoglobulin producing cells. The plate assay was validated by studying immunoglobulin production following recovery of clones from dishes and their growth to mass suspension culture. Immunoglobulin formation in these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium, and by incubating the cells with radioactive amino acids and analyzing the intracellular and secreted immunoglobulin on polyacrylamide gels.     

Cyclic AMP-mediated modulation of immunoglobulin production in B cells by prostaglandin E1.

We had previously demonstrated in a transformed human B cell line, LA350, the existence of an inverse relationship between cyclic adenosine monophosphate (cAMP) content and immunoglobulin secretion using the cAMP-elevating agents such as cholera toxin and forskolin. In this paper we report that cAMP acting as a second messenger for prostaglandin exerts a similar effect on the antibody response of B lymphocytes. Incubation of the cells with PGE1 in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) produced a concentration- and time-dependent elevation of intracellular cAMP. Significant increases of cAMP production were observed at physiologically relevant levels of PGE1 (10(-7) and 10(-8) M). Immunoglobulin production, whether measured as the total number of immunoglobulin-secreting cells by a reverse hemolytic plaque assay or as specific immunoglobulin production (IgM) by an enzyme-linked immunoadsorbent assay, was suppressed in a dose-dependent fashion by the presence of IBMX. This suppression of immunoglobulin production was significantly enhanced by the presence of PGE1. Phorbol myristate acetate-induced IgM production was also inhibited by the presence of PGE1. These results imply that prostaglandins regulate B cell activation and immunoglobulin production by signal transduction mechanisms involving cyclic nucleotides.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

Artificial chromosome vectors and expression of complex proteins in transgenic animals

Artificial chromosome vectors are autonomous, replicating DNA sequences containing a centromere, two telomeres and origins of replication. Artificial chromosomes have been proposed as possible vectors for transferring very large sequences of DNA into animals. Our goal has been to insert the entire human heavy- and light-chain immunoglobulin loci into cattle as a step in developing a production system for large quantities of human therapeutic polyclonal antibodies. A mitotically stable fragment of chromosome 14, containing the human heavy-chain locus, was identified. A chromosome cloning system was used to transfer the human lambda locus from an unstable chromosome 22 fragment to the chromosome 14 fragment to create a human artificial chromosome (HAC) carrying both immunoglobulin loci. The HAC vector was introduced into bovine primary fibroblasts. Selected fibroblast clones were rejuvenated and expanded by producing cloned fetuses. Cloned fetal cells were selected and recloned to produce 21 healthy, transchromosomic (Tc) calves. Four were analyzed and shown to functionally rearrange both heavy- and light-chain human immunoglobulin loci and produce human polyclonal antibodies. These results demonstrate the feasibility of using HAC vectors for production of transgenic livestock. More importantly, Tc cattle containing human immunoglobulin genes may be used to produce novel human polyclonal therapeutics.