Involvement of cell-cell adhesion molecules in liver colonization by metastatic murine lymphoma/lymphosarcoma variants.

Metastatic variant sublines of the murine RAW117 large cell lymphoma or lymphosarcoma have been established in vitro by sequential cycles of harvesting of liver tumor nodules after intravenous inoculation of tumor cell suspensions into syngeneic BALB/c mice. After five and ten in vivo selections for liver colonization, variant sublines RAW117-H5 and -H10, respectively, were established, and these formed significantly more surface liver tumors than the parental RAW117-P line. RAW117 sublines were tested for their abilities to adhere to embryonic mouse liver or brain cells in an in vitro cell-cell adhesion assay. Liver colonizing RAW117-H10 cells adhered with greater selectivity to liver cells than to brain cells. Parental RAW117-P cells were more homotypically adhesive, but they were nonselective in their organ cell adhesion properties. We examined RAW117 cells for the presence of liver cross-reactive antigens using polyclonal xenoantibody preparations directed against embryonic murine liver cells. These antibody preparations block organ-specific homotypic adhesion of embryonic murine liver cells in vitro. The amount of fetal liver antigen(s) expressed on RAW117 sublines correlated with liver colonization potentials (H10 greater than H5 greater than P) in quantitative absorption assays. Treatment of the highly metastatic RAW117-H10 subline with polyclonal anti-embryonic murine liver F(ab')2 or Fab' antibody fragments had no effect on RAW117-H10 cell viability or growth in vitro or in vivo, but inhibited liver colonization (median liver tumor colonies reduced from greater than 200 to 0) and prolonged life expectancy. In contrast, pretreatment of RAW117-H10 cells with polyclonal anti-H-2 did not modify the in vivo biologic properties of these metastatic cells.

Transthyretin is an inhibitor of monocyte and endothelial cell interleukin-1 production.

Human serum was found to contain an inhibitor of constitutive interleukin-1 (IL-1) production by human umbilical vein endothelial cells (ECs). Purification of the serum activity by anion exchange chromatography, molecular sieve HPLC, and hydroxyl apatite chromatography yielded material 82% pure with a molecular weight of 17 kDa by SDS-PAGE. Amino acid sequencing revealed the purified inhibitor to be transthyretin (TTR), a liver-derived protein. There was a 42.6% reduction in the production of spontaneous IL-1 activity in EC supernatants after coculture with 10 micrograms/ml TTR. TTR was subsequently found by ELISA to inhibit LPS-stimulated IL-1 production by cells of the human monocytic leukemia line THP-1 by 47.1 +/- 9.4%, whereas a less striking but still significant inhibition of monocyte-derived IL-1 beta production was also observed. Inhibition of IL-1 secretion correlated with increased IL-1 mRNA synthesis in both THP-1 cells and monocytes. Furthermore, TTR was associated with increased intracellular concentrations of IL-1 beta. These data suggest that TTR functions by inhibiting processing of newly synthesized peptide for secretion. This novel inhibitory effect of TTR on the production of IL-1 activity suggests a previously unrecognized endogenous antiinflammatory mediator.

Lymphocyte proliferation in glutathione-depleted lymphocytes: direct relationship between glutathione availability and the proliferative response.

Lymphocyte proliferation in response to mitogenic lectins is directly dependent upon glutathione (GSH) availability. Thus, proliferation can be enhanced by providing lymphocytes with excess glutathione, and strongly inhibited by limiting the quantity of intracellular GSH available during the mitogenic stimulation. Exogenous GSH, cysteine and 2-mercaptoethanol (2-ME) can all significantly enhance lymphocyte proliferation and augment intracellular GSH levels. Lymphocytes depleted of GSH by buthionine sulfoximine (BSO) fail to undergo a full blast transformation response to mitogenic lectins. In lymphocytes stimulated with mitogen in the presence of BSO, the time profile of intracellular GSH levels shows a rapid decline over the first 24-48 h and a subsequent gradual decline to levels less than 0.5 nmol/10(7) lymphocytes by 72-96 h. Exogenous GSH partially sustains intracellular GSH levels and completely restores lymphocyte proliferation even in the presence of 2000 microM BSO. Other thiols, such as cysteine and 2-ME, do not significantly alter the time profile of intracellular GSH in mitogen-stimulated lymphocytes in the presence of 2000 microM BSO, and their capacity to enhance proliferation is greatly diminished albeit not completely abolished under these conditions. Ongoing GSH synthesis is clearly essential to maintain a normal proliferative response. If intracellular GSH levels are depleted initially and lymphocytes are then stimulated with mitogen in the presence of BSO, there is a diminished capacity of cysteine and 2-ME to restore proliferation relative to exogenous GSH. There is also a diminished capacity of exogenous GSH to restore proliferation with higher concentrations of BSO. This suggests that the restoration of lymphocyte proliferation by exogenous GSH is more closely linked to effects on intracellular rather than extracellular GSH. These studies confirm the importance of intracellular GSH in lymphocyte proliferation. The essential role for intracellular GSH can be demonstrated even in the presence of other exogenous thiols, such as cysteine and 2-ME. The enhancement of lymphocyte proliferation by exogenous cysteine appears to be directly linked to effects on intracellular GSH, whereas the enhancement by 2-ME is probably more complex but clearly linked to effects on intracellular GSH.

Murine transfer factor. III. Specific interactions between transfer factor and antigen.

The interactions between dialyzable transfer factor and antigens have been studied. Incubation of transfer factor-containing dialysates from ferritin-sensitized mice or ferritin-coated plastic surfaces removed the antigen-sensitizing activity; incubations of the same preparations on cytochrome c-coated surfaces did not. Similar results were obtained when cytochrome c-transfer factor was studied. Incubation on cytochrome c-coated surfaces removed the activity, but incubation on ferritin-coated surfaces did not. Specific transfer factor activities could be recovered by elution with 8 M urea or acetonitrile. The finding of interactions between transfer factor and antigens provides evidence for a molecular basis of the specificity of the immunologic effects of transfer factor. This technique may also enable us to obtain amounts of specific material that are adequate for chemical analysis.

Anti-inflammatory effects of theophylline: modulation of cytokine production.

BACKGROUND: The basis for the efficacy of theophylline in the treatment of asthma remains enigmatic. Although commonly classified as a bronchodilator, its ability to dilate smooth muscle is considered fairly poor and clinical responses are often independent of bronchodilation. Recent studies have suggested that immunomodulatory activities may contribute to the therapeutic benefit mediated by theophylline. OBJECTIVE: We performed these preliminary studies to determine whether theophylline modulates cytokine production by peripheral blood mononuclear cells. METHODS: Peripheral blood mononuclear cells were obtained from 24 asthmatic subjects and were left in a resting state or stimulated with either mitogens (phytohemagglutinin, lipopolysaccharide) or antigen (tetanus, cat) with or without the additional presence of theophylline (15 micrograms/dL). Supernatants were collected and evaluated for cytokine concentration by ELISA. RESULTS: Theophylline neither inhibited production of allergenic cytokines such as IL-4 nor modulated the repertoire of cytokines produced by TH cells. A statistically significant inhibition of spontaneous interferon-gamma synthesis was observed (24.5 +/- 8.6 to 13.4 +/- 4.2; P < .05). Theophylline did have anti-inflammatory effects on cytokines primarily produced by mononuclear phagocytic cells. Theophylline mediated a slight inhibition of TNF-alpha production (0.26 +/- 0.08 to 0.21 +/- 0.06; P < .05). Theophylline was also associated with a 2.8-fold increase in spontaneous production of the anti-inflammatory cytokine IL-10 (0.35 +/- 0.08 to 0.98 +/- 0.16 ng; P < .01). CONCLUSIONS: A relative absence of IL-10 characterizes the asthmatic airways and may contribute to the development and severity of allergic inflammation. Induction of IL-10 production by theophylline may therefore mitigate inflammation and contribute to the clinical efficacy of this class of medications.

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.

The role of glutathione in lymphocyte activation--II. Effects of buthionine sulfoximine and 2-cyclohexene-1-one on early and late activation events.

Depletion of intracellular glutathione (GSH) inhibits the lectin-induced activation response of human T lymphocytes. GSH-depleted lymphocytes undergo a partial activation response to lectins but fail to undergo blast transformation. Several lines of evidence indicate that the inhibition of lymphocyte activation in GSH-depleted lymphocytes involves relatively late activation events. Firstly, lectin stimulation induces significant 14C-AIB uptake, IL-2 production and expression of IL-2 receptor but a near complete inhibition of 3H-uridine and 3H-thymidine incorporation. Comparable levels of IL-2 production and IL-2 receptor expression are seen in GSH-depleted lymphocytes allowed to recover from GSH depletion during lectin stimulation. However, in the latter case, 3H-uridine and 3H-thymidine incorporation are normal, and activation is completely restored. Exogenous IL-2 cannot restore activation in GSH-depleted lymphocytes. Furthermore, lymphocytes remain highly susceptible to inhibition by GSH depletion even after 48 h of lectin stimulation which is sufficient to induce early activation events in the Go----G1 transition, such as IL-2 receptor expression and IL-2 production. Exogenous GSH partially restores intracellular GSH levels and completely restores lymphocyte activation in GSH-depleted lymphocytes. Despite comparable degrees of GSH depletion, DL-buthionine-SR-sulfoximine and 2-cyclohexene-1-one inhibit lymphocyte activation to different degrees. The inhibition by 2-cyclohexene-1-one is consistently greater than would be predicted based on glutathione depletion per se. We conclude that GSH-dependent processes are important in relatively late steps of the activation sequence characterized by nuclear events with relative sparing of essential early steps in activation, such as IL-2 receptor expression and IL-2 production. The approximate minimal intracellular GSH concentration necessary to sustain a normal activation response is 2 nmol per 10(7) lymphocytes.

Murine transfer factor. II. Transfer of delayed hypersensitivity to synthetic antigens.

Synthetic polyaminoacid antigens were used to examine the specificity of transfer of delayed-type hypersensitivity with spleen cell dialysates in mice. Dialysates from GAT10-sensitized donors sensitized recipients to GAT10, but not GLA5 or cytochrome c. Dialysates from GLA5-sensitized donors sensitized recipients to GLA5, but not GAT10 or cytochrome c. We interpret these findings as consistent with the concept that passive transfer of delayed hypersensitivity with dialyzable materials is an immunologically specific event.

Regulation of low affinity IgE receptor (CD23) expression on mononuclear phagocytes in normal and asthmatic subjects.

Mononuclear phagocytic cells contain low affinity receptors for IgE (Fc epsilon RII or CD23) which induce cellular activation in the presence of specific allergen. These studies were performed to quantify the expression by monocytes and alveolar macrophages of Fc epsilon RII in asthma and to determine biologic response modifiers that regulate Fc epsilon RII. Whereas 2.5 +/- 1.0% of the monocytes obtained from normal volunteers were Fc epsilon RII positive, this increased to 16.7 +/- 2.4% in asthma (p < 0.001). Stimulation of Fc epsilon RII expression on monocytes was shown to be an activity of IL-4 (24.5 +/- 5.9%), granulocyte-macrophage-CSF (28.1 +/- 5.2%), IFN-alpha (15.8 +/- 5.3%), IFN-gamma (10.4 +/- 3.7%), and macrophage-CSF (7.3 +/- 0.7%) but not of IL-2, IL-6, or TNF-alpha. Expression of Fc epsilon RII by these cytokines was associated with the induction of specific mRNA transcripts. Using Fc epsilon RII subtype specific primers in the polymerase chain reaction expansion of cDNA, cytokine-induced receptors were shown to be Fc epsilon RIIb. Alveolar macrophages from nonasthmatic subjects displayed minimal expression of Fc epsilon RII (3.2 +/- 1.2%); however, these receptors were present on 69.2 +/- 6.3% of asthmatic volunteers (p < 0.001). Induction of Fc epsilon RII appears specific for allergic asthma insofar as these receptors are also not expressed in subjects with interstitial lung disease (1.3 +/- 1.3%). As assessed by shift in mean fluorescence, instillation of allergen in the asthmatic's airway further up-regulated Fc epsilon RII on alveolar macrophages by 151 +/- 7%. Up-regulation of Fc epsilon RII in atopic individuals may therefore reflect allergen-induced exposure of mononuclear phagocytes to one or more of these cytokines. These studies suggest a mechanism by which an immunologic stimulus that leads to the production of these cytokines (e.g., allergen or viral infection) would contribute to the development or exacerbation of allergic disease.

Sézary syndrome with elevated serum IgE and hypereosinophilia: role of dysregulated cytokine production.

A 62-year-old man presented with a 4-year history of a pruritic erythematous rash. Initial workup was not diagnostic, and the rash was refractory to standard treatment. A complete blood cell count demonstrated a white cell count of 9100 cells/microliter with 61% polymorphonuclear neutrophils, 16% eosinophils, and 17% lymphocytes. A serum protein electrophoresis revealed an M spike identified as IgG kappa. His IgE level was 11,900 IU/ml. A bone marrow biopsy specimen demonstrated "atypical plasma cells" but was not diagnostic for myeloma. Peripheral blood smear was remarkable for highly convoluted lymphoid cells diagnostic for Sézary T cells. Consistent with this diagnosis, 89% of his peripheral blood CD2+ cells expressed CD4. The eosinophilia, elevated IgE level, and monoclonal gammopathy led to further investigations. Circulating adherent monocytes were 96% positive for presumed low-affinity IgE receptor expression as shown by surface IgE binding. In a 9-day lymphocyte coculture, the patient's T lymphocytes induced IgE production in vitro (1.25 ng/ml) by a normal donor's cultured B cells. A healthy donor's T cells failed to induce IgE production (0 ng/ml) in a similar culture system. In situ hybridization with an Sulfur-35-labeled cDNA probe revealed interleukin-4 mRNA expression by 84% and interleukin-2 mRNA expression by 62% of nonadherent peripheral blood mononuclear cells. Interleukin-5 mRNA was shown by reverse transcription and the polymerase chain reaction. These studies demonstrate a subject with Sézary T cell leukemia with hypereosinophilia and elevated IgE due to presumed enhanced interleukin-4 and interleukin-5 production by the Sézary T cells.

Anti-inflammatory effects of nedocromil sodium: inhibition of alveolar macrophage function.

The IgE-dependent activation of mononuclear phagocytic cells through their capacity to express low affinity IgE receptors (Fc epsilon RII) has been proposed as a mechanism for the development of airways inflammation in allergic asthma. This Fc epsilon RII expression leads to the IgE-dependent production of the potent pro-inflammatory cytokines IL-1 beta and TNF-alpha. Expression by monocytes of Fc epsilon RII is regulated by several cytokines including interleukin-4, gamma- and alpha-interferons, and granulocyte-macrophage and macrophage colony stimulating factors. An anti-inflammatory effect of nedocromil on monocytes has been proposed as a possible mechanism for its anti-asthma activity. We therefore investigated the capacity of nedocromil to modulate mononuclear phagocyte Fc epsilon RII expression and cytokine production. We used an anti-Fc epsilon RII antibody and flow cytometric analysis to assess the capacity of nedocromil to modulate cytokine-induced Fc epsilon RII expression in normals and asthmatics. Monocytes, THP-1 monocyte leukaemia cells, and alveolar macrophages were exposed to varying concentrations of these cytokines for 48 hr at 37 degrees C with or without the additional presence of nedocromil (1-10 microM) and the per cent of monocytes expressing Fc epsilon RII was determined. No changes in Fc epsilon RII expression were observed. Subsequently, we investigated the capacity of nedocromil to affect the capacity of IgE plus anti-IgE complexes, allergen, and LPS (16 hr/37 degrees C) to stimulate IL-1 beta and IL-6 production. No changes were observed when nedocromil was applied concomitant with the stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of dendritic cells as stimulators of minor lymphocyte-stimulating locus-specific T cell responses in the mouse. I. Differential capacity of dendritic cells to stimulate minor lymphocyte-stimulating locus-reactive T cell hybridomas and the primary anti-minor lymphocyte-stimulating locus mixed lymphocyte reaction.

The response of T cells to minor lymphocyte-stimulating locus (Mls) determinants remains poorly understood with respect to the antigenic determinants responsible for T cell stimulation and the types of APC capable of stimulating the response. In this report, we demonstrate that highly purified dendritic cells (DC) as well as B cells have the capacity to stimulate Mls-specific responses. Unseparated spleen cells, purified DC, resting B cells, and activated B cells were compared for their capacity to stimulate several Mls-reactive T cell hybridomas. Whereas the entire panel of Mls-reactive T cell hybridomas was stimulated strongly by unseparated spleen cells and activated B cells, the hybridomas responded only weakly to purified DC or resting B cells. Activation of resting B cells with either B cell stimulatory factor-1 (1 day pre-treatment) or LPS/dextran (2 or 3 day pre-treatment) greatly augmented their Mls-stimulatory capacity. In contrast, the Mls-stimulatory capacity of DC was not augmented by a 1-day pre-treatment with either B cell stimulatory factor-1 or supernatant from the DC-induced primary anti-Mls-MLR. In the primary anti-Mls-MLR, both purified DC and LPS/dextran-stimulated B blasts were found to elicit vigorous T cell proliferative responses. Much weaker responses were elicited by unseparated spleen cells. The stimulation of the primary anti-Mls-MLR by purified DC was further confirmed by producing Mls-specific T cell clones which were preferentially stimulated by DC. Autologous (Mlsb) DC were found to markedly enhance the primary anti-Mls-MLR response to small numbers of Mlsa B blasts. Thus, DC possess other "accessory cell" properties that augment the primary anti-Mls-MLR despite the predicted low level of Mls determinant expression on DC based on the results obtained with Mls-reactive hybridomas. Possible accessory cell properties of DC relevant to this phenomenon are discussed.

Detection of alveolar macrophage-derived IL-1 beta in asthma. Inhibition with corticosteroids.

This study investigated alveolar macrophage function in asthma as assessed by the presence and source of the cytokine IL-1 beta. Bronchoalveolar lavage (BAL) fluid and cell pellets were obtained at 4 a.m. from a cohort of asthmatic subjects. Normal subjects were used as controls. Asthmatic BAL fluid contained 57.0 +/- 5.9 pg/ml of IL-1 beta as determined by ELISA. No IL-1 beta could be identified in the BAL fluid of the control group. This IL-1 represented synthesis by cells of the airway as assessed by the presence of IL-1 beta-specific mRNA transcripts in the BAL cell pellets. Inasmuch as IL-1 beta transcripts are known to be present for only a short time period after activation (approximately 0.5 to 16 h), the presence of transcripts represents recent cellular activation. Using the technique of in situ hybridization, IL-1 beta transcripts were found to be located exclusively within alveolar macrophages, and a mean of 40 +/- 14% of alveolar macrophage were activated at the time of the lavage. Corticosteroids are known to be efficacious in the treatment of asthma. Administration of a single 50-mg dose of prednisone at 3 p.m. resulted in diminished IL-1 beta protein concentration in the BAL fluid (28.3 +/- 5.7 pg/ml; p < 0.01 compared to baseline) and completely abrogated IL-1 beta mRNA expression. In summary, these studies demonstrate that IL-1 beta is synthesized within the airways by alveolar macrophage and suggest that IL-1 beta may be a mediator of asthma. Inhibition of IL-1 beta may be an additional mechanism for the efficacy of corticosteroids in the treatment of asthma.