Mouse Peritoneal Exudate Cells Research Articles

Factors affecting the escape of Francisella tularensis from the phagolysosome.

The highly virulent bacterium Francisella tularensis is well adapted to the intracellular habitat but the mechanisms behind its intracellular survival have been elusive. Recently, it was shown that the bacterium is capable of escaping from the phagosome of human and mouse monocytic cells. Here it is shown that this escape is affected by gamma interferon (IFN-gamma) treatment of mouse peritoneal exudate cells since in treated cells the proportion that escaped was significantly lower (80%) than in untreated cells (97%) as determined by transmission electron microscopy. By contrast, < 1% of mutant bacteria lacking expression of a 23 kDa protein denoted IglC were able to escape from the phagosome. Infection with the DeltaiglC strain complemented with the iglC gene resulted in 60% of the bacteria escaping from the phagosome. Whereas IFN-gamma treatment conferred a static effect on intracellular wild-type bacteria, the treatment had a bactericidal effect on the DeltaiglC strain. The results show that the activation status of infected cells affects the escape of F. tularensis from the phagosome. An even more profound effect on this escape is related to expression of IglC by F. tularensis. Its absence rendered the mutant bacteria incapable of escaping from the phagosome and of multiplying intracellularly.

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.

Study on the effect of polybutyl-2-cyanoacrylate nanoparticles and their metabolites on the phagocytic activity of peritoneal exudate cells of mice

The phagocytic activity of peritoneal exudate cells (PECs) harvested from peritoneal cavity of mice after a single intraperitoneal (i.p.) treatment with poly(butylcyanoacrylate) nanoparticles (PBCN) and their probable metabolites [poly(cyanoacrylic acid) (PCAA) and n-butanol] was investigated in an in vitro phagocytic assay. Polymer suspension of PBCN was given as a single i.p. injection at doses of 200 and 10 mg kg −1, 3, 18, 72 and 120 h before the performance of the phagocytic assay. PCAA and n-butanol were given at the same manner at doses of 126.8 and 96.8 mg kg −1, respectively (equivalent to a dose of 200 mg kg −1 of intact PBCN after enzyme hydrolysis) 3, 18 and 120 h before the test performance. The phagocytic assay was performed in vitro in tubes with sheep red blood cells (SRBC). Phagocytic index (percentage of PECs ingested more than 3 sheep erythrocytes), phagocytic number, and ingestion capacity (number of erythrocytes ingested per cell) were the parameters used for evaluation of the phagocytic activity. The alterations of phagocytic activity of the PECs observed were strongly time- and dose-dependent. Administration of all tested compounds shortly before the test performance resulted in a considerable decrease in the capability of PECs to ingest SRBC. The alterations of phagocytic activity decreased when the time between the treatment of mice and the phagocytic assay is on the increase. The dose of 200 mg kg −1 of PBCN administered 120 h before the phagocytic assay led to the significant increase of the phagocytic index of PECs. The phagocytic function of assayed PECs was temporary impeded and 5 days were completely enough for their restoration.

Heparin-Binding Protein Decreases Apoptosis in Human and Murine Neutrophils

Background. Heparin-binding protein (HBP), a serine protease without any known proteolytic activity, is found in human polymorphonuclear leukocyte (PMN) granules, but not in mice. HBP potentiates the endotoxin-induced release of tumor necrosis factor (TNF) α, interleukin (IL)-1, and IL-6 from isolated monocytes. HBP has also been shown to increase the survival of cultured monocytes and protect them from oxidative stress. However, whether HBP affects PMNs themselves is not known.Materials and Methods. Based on our work with cultured monocytes and the survival benefit noted in experimental peritonitis, we hypothesized that HBP would have a beneficial effect on the survival of neutrophils. We evaluated the effect of HBP on apoptosis in murine peritoneal exudative cells elicited by intraperitoneal thioglycollate administration and in normal human neutrophils from volunteers. Leukocytes were isolated from the peritoneal cavity and blood of mice that underwent intraperitoneal thioglycollate instillation. The mouse peritoneal exudate cells and normal human neutrophils isolated from peripheral blood were used to study the effect of HBP on survival and apoptosis.Results. HBP decreased percentage apoptosis of mouse cells in both serum-enriched (from 24.8 to 4.5%) and serum-deprived (from 23.1 to 8.2%) cultures. In human PMNs, the protective effect of HBP was seen only in the serum-deprived group, with a decrease in percentage apoptosis from 69.1 to 43.3%.Conclusions. For the first time, we have shown that HBP, in addition to its known augmentation of the proinflammatory response of monocytes, also acts as a prosurvival protein for neutrophils themselves, and thereby enhances local host defense.

抗酸菌に対する予防ワクチン

Studies on recombinant BCG (rBCG) which my group carried out so far were reviewed. Recombinant BCG which secreted alpha antigen-fused foreign antigen was constructed and tested for its ability to induce protective immunity. Thus, rBCG secreting merozoite surface protein 1 (MSP1) of Plasmodium yoelii efficiently protected the infection more than recombinant MSP 1 mixed with artificial adjuvant RAS or IFA did. rBCG which secreted excess amounts of antigen 85 complex A inhibited the multiplication of M. leprae in the footpads of mice. rBCG which secreted alpha antigen-fused IL-2 stimulated peritoneal exudate cells of mice resulting in enhancing killing a bladder cancer cell line in vitro.

Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide

The pleiotropic cytokine interleukin-1 (IL-1) is an inducer of the inducible nitric oxide synthase (iNOS). It was surprising to find that treatment of normal mice with an iNOS inhibitor resulted in detectable IL-1beta mRNA in colon and spleen, suggesting feedback regulation. When mouse peritoneal exudate cells (PEC) or RAW 264.7 cells were stimulated with lipopolysaccharide (LPS), concomitant inhibition of iNOS resulted in an increase of IL-1beta and IL-1alpha protein secretion. Conversely, after addition of the NO-generating compound NOC-18, IL-1beta and IL-1alpha concentrations in supernatants were dose-dependently reduced. Costimulation with interferon-gamma (IFN-gamma) reversed the NOC-18-mediated suppression of IL-1alpha protein concentration into an almost fivefold increase in RAW 264.7 cells. This effect was specific for IL-1alpha and was also seen in PEC. The mRNA expression for IL-1beta and IL-1alpha in RAW 264.7 cells correlated with the protein levels, suggesting transcriptional regulation by NO. Dysregulated IL-1/NO cross-regulation may play a role in inflammatory diseases.

Role of TNF-α in the Induction of Fungicidal Activity of Mouse Peritoneal Exudate Cells againstCryptococcus neoformansby IL-12 and IL-18

We have recently demonstrated that two IFN-γ-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) againstCryptococcus neoformansthrough NK cell production of interferon (IFN)-γ and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-α in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-α mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-γ production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-α by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-γ mAb. The cell type producing TNF-α among PEC was mostly macrophage. TNF-α significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-γ, and furthermore anti-TNF-α mAb partially inhibited these responses. Considered together, our results indicated that TNF-α contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity againstC. neoformansthrough enhancement of IFN-γ-induced production of NO by macrophages, but not through increased production of IFN-γ by NK cells.

In vivo priming of mice with antigen and lipid A bestows proliferative ability on peritoneal exudate T cells in response to antigen or anti-αβ TCR antibody in the absence of macrophages in vitro: accessory function of NK cells

Highly purified T cells of mice that have been primed in vivo with horse red blood cells (HRBC) and lipid A, but not with HRBC alone, can proliferate in vitro in response to HRBC or anti-αβ T cell receptor (TCR) antibody without macrophages. The mechanism of proliferation without macrophages was investigated. Mice were primed with HRBC and lipid A together or with HRBC alone 10 days in advance, and the peritoneal exudate cells (PEC) were obtained. Purified T cells were obtained by treating the PEC by the following series of procedures: nylon fiber column-passage, antibody (anti-Ia, anti-Mac-1, and LR-1) treatments with complement (C), and a G-10 column-passage. Cell proliferation was assessed by [3H]-thymidine (TdR) incorporation into the cultured T cells in response to HRBC or anti-αβ TCR antibody. The proliferation of T cells [T(HRBC+lipid A)] that had been prepared from the PEC of the mice primed with HRBC and lipid A increased dose-dependently to these stimulants, but T cells [T(HRBC)] prepared from PEC of mice primed with HRBC showed little or no proliferation in response to them. The expression pattern of a memory cell marker, CD44, on the cell surface of T(HRBC+lipid A) was obviously different from that on T(HRBC). The proliferation of T(HRBC+lipid A) was suppressed when the cells were cultured in the wells coated with hyaluronate, a ligand for CD44, or cultured after a previous treatment with anti-CD44 (IgG2a) and anti-IgG2a antibodies. In contrast, the proliferation of T(HRBC) was up-regulated in culture under the same conditions. Proliferative responses of T(HRBC+lipid A) to anti-αβ TCR antibody were enhanced in the wells coated with anti-CD28 antibody. These findings indicate that the proliferation of T(HRBC+lipid A) was not only supported by a stimulation of TCR but also regulated by another stimulation via ligands such as CD44 and CD28. The proliferation of T(HRBC+lipid A) was abolished when the cells were pretreated with AsGM1 antibody or anti-CD80 antibody and C. These findings indicate that the in vivo priming of mice with HRBC and lipid A generates a memory T cell population that is capable of proliferating without help of macrophages, but with NK cells, when stimulated their TCR.

Combined effects of IL-12 and IL-18 on the clinical course and local cytokine production in murine pulmonary infection with Cryptococcus neoformans

We reported recently that interleukin (IL)-12 and IL-18 synergistically increased the fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by inducing the production of interferon (IFN)-γ by natural killer (NK) cells. To confirm these findings in vivo, we examined the effect of combined treatment using these two cytokines on the course of experimentally induced pulmonary and disseminated cryptococcosis in mice. IL-12 andIL-18 were used at subtherapeutic doses (0.005 and 2 μg/mouse/day, respectively). A single administration of either cytokine was not effective in protecting mice against the infection, while combined treatment significantly prolonged survival time of infected mice and reduced the lung and brain loads of organisms. These protective effects were associated with elevated IFN-γ and reduced IL-4 levels in bronchoalveolar lavage fluid. Finally, depletion of NK and γ δ T cells, but not of CD4+ T cells, by administration of specific antibodies, significantly reduced the production of IFN-γ in lungs by IL-12/IL-18 treatment during the 7 days of infection. Our results demonstrated that IL-12 and IL-18 protected mice against cryptococcal infection in a synergistic manner by enhancing the local production of IFN-γ by NK and γ δ T cells in the early phase of infection and by suppressing the production of IL-4 in lungs.

Combined effects of IL-12 and IL-18 on the clinical course and local cytokine production in murine pulmonary infection with Cryptococcus neoformans.

We reported recently that interleukin (IL)-12 and IL-18 synergistically increased the fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by inducing the production of interferon (IFN)-gamma by natural killer (NK) cells. To confirm these findings in vivo, we examined the effect of combined treatment using these two cytokines on the course of experimentally induced pulmonary and disseminated cryptococcosis in mice. IL-12 and IL-18 were used at subtherapeutic doses (0.005 and 2 microg/mouse/day, respectively). A single administration of either cytokine was not effective in protecting mice against the infection, while combined treatment significantly prolonged survival time of infected mice and reduced the lung and brain loads of organisms. These protective effects were associated with elevated IFN-gamma and reduced IL-4 levels in bronchoalveolar lavage fluid. Finally, depletion of NK and gammadelta T cells, but not of CD4+ T cells, by administration of specific antibodies, significantly reduced the production of IFN-gamma in lungs by IL-12/IL-18 treatment during the 7 days of infection. Our results demonstrated that IL-12 and IL-18 protected mice against cryptococcal infection in a synergistic manner by enhancing the local production of IFN-gamma by NK and gammadelta T cells in the early phase of infection and by suppressing the production of IL-4 in lungs.

Human IL-6 Enhances Human Lymphocyte Engraftment and Activation but not Human Antibody Production in SCIDhu PBL Mice

The SCIDhu PBL model of human Ig production was modified by using human interleukin-6 (hIL-6) secreting tumors for continuous hIL-6 production, in vivo. On day one, SCID mice were injected subcutaneously with 200 µl PBS (control mice), 10 4 SP2/0-Ag14 cells (IL-6 − mice) or 10 4 hIL-6 secreting SP2/0-hIL6.17 cells (IL-6 + mice). The mice were reconstituted with human PBMC on day two and immunized with 100 µg of tetanus toxoid (TT) on days two and fifteen. Serum hIL-6 concentrations in IL-6 + mice ranged between 2.9 and 38.1 ng/ml by days 26−33. IL-6 + mice had enlarged spleens and lymph nodes (LN). Flow cytometry and histology showed that SCIDhu PBL mouse spleen, LN and peritoneal exudate cells (PEC) contained mostly murine myeloid lineage cells. In addition, many more human B cells, T cells and IL-2R +-activated lymphocytes were present in spleen, LN and PEC of IL-6 + mice. Despite enhanced lymphocyte engraftment and activation, by day 14 IL-6 + mice produced up to 6-fold less TT-specific IgG relative to total IgG than either control group. TT-specific and total Ig sera concentrations were equivalent in all three groups on days 26–33. Our results suggest that sustained circulating hIL-6 enhanced human delayed-type hypersensitivity (DTH)-like inflammatory responses with consequential inhibition of TT-specific IgG production in SCIDhu PBL mice.

Differential regulation of macrophage arginine metabolism: a proposed role in wound healing

Nitric oxide (NO) and ornithine, products of NO synthase or arginase, respectively, have opposing biological activities. The effect of mediators of leukocyte activation and inhibition on arginine metabolism of resident mouse peritoneal exudate cells (MPEC) was determined. Factors that increased basal NO synthase activity, interferon (IFN)-gamma and lipopolysaccharide (LPS), decreased arginase activity in intact cells. Transforming growth factor (TGF)-beta1 decreased IFN-gamma-stimulated NO synthase activity and produced a reciprocal increase in urea and ornithine release. TGF-beta1 had no effect on the activity of these enzymes in LPS-stimulated MPEC. Corticosterone (Cort, 100 ng/ml) decreased the basal activity of both enzymes. However, Cort inhibited NO synthase activity and increased ornithine release in MPEC exposed to IFN-gamma or LPS. The difference between arginase activity in intact cells vs. that of cell lysates suggested intracellular inhibition of arginase activity. Products of NO synthase, NO and citrulline, were shown to inhibit MPEC arginase activity under maximal assay conditions. Intracellular pH was not altered by exposure of MPEC to LPS, IFN-gamma, TGF-beta, and Cort. This reciprocal change in arginine metabolism is proposed to be an important component of wound healing. Expression of NO synthase creates a cytotoxic environment that may be important to the early phase of wound healing. As wound healing progresses, increased arginase activity produces an environment favorable for fibroblast replication and collagen production.

Immunomodulation and antitumor activity of polysaccharide-protein complex from the culture filtrates of a local edible mushroom, Tricholoma lobayense

1. 1. The polysaccharide-protein complex(PSPC) isolated from the culture filtrate of Tricholoma lobayense showed antitumor activity in mice. 2. 2. PSPC had the ability to restore the phagocytic function of peritoneal exudate cells (PEC) and T-cell mitogenic activity of tumor-bearing mice. 3. 3. An increase in the production of reactive nitrogen intermediates and tumor necrosis factor (TNF) was determined in PEC of mice that had received PSPC. 4. 4. The present study showed that PSPC both induced the various immune responses in vivo and exhibited cytotoxicity against tumor cell lines in the presence of PSPC at doses of 30 and 60 μ/ml in vitro.

Experimental investigations on the immunomodulating activity of polybacterial preparation for peroral immunotherapy and immunoprophylaxis of uroinfections

The immunomodulating effect of a polybacterial immunostimulator Urostim in experimental animal models was investigated. The preparation was created for immunotherapy and immunoprophylaxis of uroinfections. It consists of killed bacterial cells and their lysates of four microbial species: Escherichia coli expressing type 1 pili, Rc mutant of E. coli, Klebsiella pneumoniae, Proteus mirabilis and Enterococcus faecalis, BALB/c mice and guinea-pigs were treated orally with Urostim for 5 or 10 days, respectively. A stimulatory effect of Urostim on phagocytosis (increased phagocytic index and phagocytic number) of mice peritoneal exudate cells was observed. An increased number of plaque-forming cells and elevated titres of haemagglutinating antibodies in mice demonstrated activation of humoral immunity. Lymphoproliferative studies showed a significant response to phytohaemagglutinin (PHA) and Urostim, especially after the second application of the preparation. At the same time no changes in the absolute number of peripheral blood lymphocytes were found. An active protection of mice against a systemic infection and of guinea-pigs against uroinfection caused by Gram-negative bacteria was obtained. In conclusion, the results obtained characterize Urostim as an effective immunostimulator. Its peroral application leads to the activation of innate resistance, cell-mediated, humoral and local immunity.

Analysis of antitumor properties of effector cells stimulated with a cell wall preparation (WPG) of Bifidobacterium infantis.

Intestinal Bifidobacterium species are thought to be beneficial in animal and human intestines. We studied the mechanisms of Bifidobacteria in antitumor activity using a cell wall preparation (WPG) of B. infantis (Cancer Res., 45, 1300, (1985)). WPG enhanced the in vitro antitumor activities of mouse peritoneal exudate cells elicited with proteose-peptone (P-PEC) and thioglycollate broth (TG-PEC), determined by cytostatic ([3H]thymidine uptake inhibition) and cytolytic ([3H]uridine release) assays. Tumor necrosis factor-alpha (TNF-alpha) and reactive nitrogen intermediates (RNI) play a role in such augmented cytotoxicity, because anti-TNF-alpha antibody almost completely blocked the increased cytolytic activity of P-PEC in the presence of WPG. Moreover, WPG induced RNI in the supernatant of TG-PEC in a dose-dependent manner. The mRNA expression of several cytokines (IL-1 beta, IL-6, IL-10, IFN-alpha and TNF-alpha) was induced in BALB/c mouse peritoneal cells 3 h after an intraperitoneal injection of WPG (3 h WPG-PEC). However, this expression disappeared from 24 h WPG-PEC, except for that of IFN-alpha. IFN-gamma was not induced. Kinetic studies of the tumor neutralizing activities of the WPG-PECs by means of the in vivo Winn assay revealed that the activity emerged at 1.5 h, became maximal at 3 h and disappeared at 24h. These results indicated that Bifidobacterial WPG is a Biological Response Modifier (BRM) with characteristics similar to those of other bacterial BRMs.

In vitro antiphagocytic effect of Melia azedarach leaf extracts on mouse peritoneal exudate cells

The effect of Melia azedarach L. (Meliaceae) leaf extract on the phagocytic capability and respiratory burst of mouse peritoneal exudate cells was studied. The extract inhibited the phagocytosis of opsonized sheep erythrocytes. This inhibition was both dose- and time-dependent and reverted 48 h after removing the extract from the culture medium. Furthermore, chemiluminescence in treated cells was also impaired using either receptor (opsonized zymosan) or postreceptor (PMA) stimuli.

Macrophage properties during peripheral nervous tissue rejection in vitro.

The present study introduces an in vitro model of xenogeneic peripheral nerve rejection to analyze the role of macrophages in this complex immunological situation. Nerve-sensitized mouse peritoneal exudate cells were co-cultured with rat peripheral nerve segments. The cultured rat nerve segments were fulminantly rejected in vitro by the co-cultured mouse peritoneal cell population. The massive tissue destruction included Schwann cell damage and was quite distinct from basic myelin phagocytosis observed during Wallerian degeneration in earlier experiments. The nerve-sensitized peritoneal exudate consisted of macrophages and T-cells. Antibody depletion experiments were performed to analyze T-lymphocyte effects in this model. Fulminant tissue rejection depended on the presence of T-lymphocytes in the culture medium. Their presence at the immediate site of tissue rejection, however, was not required. Further experiments were aimed at defining the role of T-cell-derived mediators during in vitro rejection. Depletion experiments using a panel of antibodies to cytokines revealed a critical involvement of IL-2, IL-3, IL-4, IL-6 and interferon-gamma in the induction of tissue rejection in vitro. Fulminant tissue rejection in vitro depended on the interaction of these cytokines with macrophages. The participation of macrophage surface receptors was studied in another series of experiments. The macrophage complement receptor type 3 was shown to be critically involved in the phagocytic attack during rejection. Antibodies to MHC class II antigens also abolished fulminant in vitro rejection, indicating that continuous antigen presentation is required in this process.

腹腔内細胞のグルコース消費能および貪食能に及ぼすケフィール粒水溶性画分の影響

腹腔内細胞のグルコース消費能および貪食能に及ぼすケフィール粒水溶性画分の影響

In vitro and in vivo induction of tumor necrosis factor alpha by Borrelia burgdorferi.

Tumor necrosis factor alpha (TNF-alpha) is an immunoregulatory cytokine with many biological activities including the mediation of inflammation. We examined sera and synovial fluids from patients seropositive for infection with Borrelia burgdorferi using a radioimmunoassay specific for TNF-alpha. Significant elevation of TNF-alpha was found in the sera and synovial fluids of patients examined, while controls showed no elevation. Sera of mice infected with B. burgdorferi contained elevated levels of TNF-alpha which varied during the course of a 24-day infection. To determine whether B. burgdorferi is capable of inducing TNF-alpha production, spirochetes were added to adherent human peripheral blood mononuclear cells or mouse peritoneal exudate cells and 24 h later supernatants were assayed. TNF-alpha induction occurred in a dose-dependent manner. The maximum stimulation occurred when a ratio of 1 to 10 spirochetes per mononuclear cell was used. At optimal concentrations, induction was not diminished by inactivation of spirochetes or pretreatment with polymyxin B. These results suggest that an increase in TNF-alpha production may occur as a result of infection with B. burgdorferi.

Enhancing effect of mouse peritoneal exudate cells and their products on antibody productivity of hybridoma cells: application of in vivo factors to in vitro culture.

Mouse peritoneal exudate cells induced by casein enhanced in vitro antibody production rate per cell of a hybridoma in co-culture. Culture supernatant of the exudate cells also enhanced three-fold the antibody productivity when added to cultures of a hybridoma at 10% (v/v). Hence the enhancement of antibody productivity by the exudate cells seemed to be caused by soluble enhancing factors secreted by the exudate cells. The exudate cells maximally secreted the enhancing factors when harvested from mice on day 4 of the induction period following the injection of casein. A semi-continuous culture of the hybridoma demonstrated the applicability of the culture supernatant to enhance antibody production by producing a two-fold increase over the control for seven days when supplemented with the supernatant at 5%. Significant amounts of interleukin-6 were detected in culture supernatant of the exudate cells. Interleukin-6 obtained from other sources enhanced the antibody productivity two-fold when added to the hybridoma culture at the concentration of 5 unit/ml. Interleukin-6, therefore, is expected to be one of the principal antibody enhancing factors secreted by the exudate cells. Other interleukins examined, that is, interleukin-1 to -5 did not enhance the antibody productivity.