Human intravenous immunoglobulin modulates monokine production in vitro.

The effects of human immunoglobulin preparations for intravenous use (IVIg) on in vitro-induced monokine production were studied. Individual peripheral blood monocytes, obtained from healthy blood donors, which produced interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) after in vitro stimulation, were identified by cytokine-specific monoclonal antibodies (mAb) and indirect immunofluorescence technique. Lipopylosaccharide (LPS) or Borrelia burgdorferi spirochetes (Bb) were used to induce TNF-alpha and IL-6 production in cultures. Peak synthesis occurred 2.5 hr after initiation of the cultures in the majority of the monocytes, but not at all in lymphocytes. The monocytes were identified by two-colour staining using a monocyte-specific mAb. IL-6 was produced by 64 +/- 8% or 71 +/- 9% (means +/- SD) of the non-IVIg-exposed monocytes after LPS or Bb stimulation, respectively (n = 12). A dose-dependent and significant reduction of the number of IL-6-producing cells was noted in the IVIg-supplemented cultures (P less than 0.003). In these cultures 24 +/- 12% or 29 +/- 12% of the monocytes made IL-6 in response to LPS or Bb. Kinetic studies indicated a sustained significant inhibition of IL-6 production during 24 hr of culture (P less than 0.001). In contrast, TNF-alpha synthesis was not inhibited by IVIg. LPS or Bb stimulation resulted in 47 +/- 18% or 69 +/- 7% TNF-alpha producing cells versus 48 +/- 9% or 59 +/- 8% in IVIg-supplemented cultures. These results indicate down-regulation of IL-6, but not TNF-alpha production, by IVIg. A direct antigen neutralization is an unlikely explanation for the divergent effects observed on monokine production after IVIg addition.     

Suppression of immunoglobulin production of lymphocytes by intravenous immunoglobulin

The proliferative responses and the immunoglobulin production of peripheral blood mononuclear cells to pokeweed mitogen were dose-dependently suppressed by sulfonated intravenous immunoglobulin (IVIG), polyethylene glycol-treated IVIG, pH 4-treated IVIG, or human -globulin, but they were not or only slightly suppressed by human serum albumin or pepsin-treated IVIG. Moreover, the suppression of immunoglobulin production by sulfonated IVIG, polyethylene glycol-treated IVIG, or pH 4-treated IVIG was seen in the cases in which B cells preincubated with IVIGs were cocultured with T cells and monocytes preincubated with or without IVIGs and in the cases in which monocytes preincubated with IVIGs were cocultured with T cells and B cells preincubated with or without IVIGs. However, in the cases in which only T cells were preincubated with IVIGs, immunoglobulin production was not suppressed. The suppression of the monocyte function by IVIGs tended to be less than the suppression of the B-cell function by IVIGs. Moreover, the suppression by IVIGs was blocked by antihuman IgG Fc. Our results suggest that IVIGs suppress the immunoglobulin production of lymphocytes through suppression of the B-cell function and the antigen presenting-cell function by attachment of IVIGs to Fc receptors of B-cell membranes and antigen presenting-cell membranes.     

Modulation of immunoglobulin production and cytokine mRNA expression in peripheral blood mononuclear cells by intravenous immunoglobulin

Intravenous immunoglobulin (IVIG) has the potential to regulate Ig production, but the mechanism(s) responsible for this effect is unknown. In experiments reported here, we examined the ability of IVIG to regulate Ig production in human peripheral blood mononuclear cells (PBMCs) stimulated with pokeweed mitogen (PWM). IVIG (2–10 mg/ml) showed a potent (80–85%) inhibition of PWM-stimulated IgG, IgM, and IgA production. To determine more precisely how IVIG mediated the inhibition of Ig production, we studied Ig promoting cytokine gene expression after PWM stimulation with or without IVIG (2 and 10 mg/ml) using dot-blot techniques. RNA was isolated from PBMCs at predetermined time points and probed with cDNAs specific for human cytokines (IL-1-, IL-2, IL-2R, IL-4, IL-5, IL-6, -IFN, and TNF-). IL-6 mRNA accumulation was maximal at 4.5 hr post-PWM stimulation and was inhibited 64–75% when IVIG (10 mg/ml) was present. -IFN mRNA levels peaked at 72 hr poststimulation and were also 68–75% inhibited by IVIG. IL-2 mRNA levels peaked at 4.5 hr and were 23–46% inhibited by IVIG. The inhibitory effect of IVIG on production of these cytokines (IL-6 and -IFN) was also observed at the protein level in sonicated PBMCs after incubation with PWM and IVIG. The mRNA levels for other cytokines were not or only minimally inhibited by IVIG. Addition of IL-6, -IFN, or IL-2 partially restored Ig production in IVIG-treated PWM-stimulated cultures, suggesting that inhibition of other cytokines or another mechanism(s) independent of cytokine inhibition might also be involved, although inhibition of IL-6, -IFN, and IL-2 may be one of the critical factors in the suppression of Ig production by IVIG.     

Indomethacin inhibitsin vitro immunoglobulin production by human B-lymphocytes

Immunoglobulin production from human B-lymphocytes stimulatedin vitro with pokeweed nitogen (PWM) was measured after 11 days of cell culture. IgM and IgG levels in culture supernatants were quantified using ELISA techniques. Addition of the prostaglandin cyclooxygenase inhibitor indomethacin (10^–7 M) to cell cultures concurrently with PWM, inhibited both IgM and IgG synthesis by 80–90% in the four individuals studied. Reduction of immunoglobulin production was apparently concentration-related over the range studied (10^–6–10^–8 M) and was not associated with cytotoxicity. If this action of indomethacin results from inhibition of cyclooxygenase, then prostaglandins or other arachidonic acid products could be involved in the regulation of human B-lymphocyte immunoglobulin production.     

Inhibition of IgE production of mice by non-specific suppressor T cells.

SWR adult normal splenic T lymphocytes injected into irradiated syngeneic mice along with immune spleen cells can suppress IgE formation by the immune cells. Spleen cells from 1- or 3-week-old mice, however, are not effective in abrogating IgE synthesis. The T lymphocytes which are induced to become suppressors for IgE are hydrocortisone-resistant and anti-thymocyte serum-sensitive. Normal SJL mice, a poor reagin-producing strain, do not appear to have more suppressor cells or cells which are more easily stimulated to become suppressor cells than a good reagin-producing strain (SWR).     

In vitro immunoglobulin production by mononuclear cells in rheumatoid arthritis

Since patients with rheumatoid arthritis (RA) exhibit serum hypergammaglobulinemia and autoantibody (rheumatoid factor) production, we compared elaboration and control of in vitro RA mononuclear cell (MNC), Ig assayed by enzyme-linked immunoassays or by hemolytic plaque formation, in 37 RA patients and 17 normal subjects. We found (1) RA spontaneous plaque-forming cells were significantly reduced (RA 344 vs normal 627 PFC/10(6) MNC, P less than 0.002); (2) RA spontaneous IgG and IgM (but not IgA) elaboration was significantly diminished (IgG RA 339, normal 776; IgM RA 255, normal 869 ng/ml, P less than 0.001; IgA RA 87, normal 124); (3) RA stimulated IgG and IgM production (but not IgA) was also decreased (IgG RA 2434, normal 3862, P less than 0.06; IgM RA, 1676, normal 3323, P less than 0.005; IgA RA 1859, normal 2315); (4) reduced RA Ig elaboration was not clearly due to altered numbers of T or non-T cells, age, medications, clinical features of disease, or response kinetics; (5) relative improvement of RA in vitro IgG, but not usually IgM, secretion followed removal of adherent cells, addition of indomethacin or addition of mitomycin C-treated T cells; (6) MNC from synovial fluids, but not bone marrows, exhibited spontaneous Ig production in excess of stimulated synovial fluid cellular or peripheral blood Ig elaboration. These observations indicate selective impairment of peripheral blood MNC IgG and, particularly, IgM secretion in RA. This defect appears to reflect accessory cell influences which differ from normal as well as the sequestration of primed or activated cells in the synovial fluid.     

Inhibition of in vitro HBsAg production by amphotericin B and ketoconazole

The effects of amphotericin B, ketoconazole, and adenine arabinoside on production of hepatitis B surface antigen (HBsAg) particles by the human hepatoma cell line PLC/PRF/5 were examined. In addition, the effects of these drugs on cellular protein synthesis were determined. These drugs caused a dose-dependent decrease in HBsAg production that was paralleled by a decrease in cellular protein synthesis. Ketoconazole was the most active of these drugs and the most specific, causing a 72% reduction in HBsAg production with only a 38% reduction in protein synthesis. These data suggest that further studies evaluating ketoconazole for the treatment of chronic hepatitis B virus infection in animals are warranted.     

Effects of retinoids on in vitro and in vivo IgE production

BACKGROUND: Retinoids modulate the growth and number of different cell types, including B cells. We could previously show that retinoic acid (RA) strongly inhibits CD40 + IL-4-mediated IgE production in vitro. The aim of the present study was to extend these findings regarding the potential use of retinoids for the treatment of allergic diseases. METHODS: In vitro IgE production was studied in anti-CD40 + IL-4-stimulated peripheral blood mononuclear cells (PBMC) from allergic donors in the presence of 10(-15)-10(-5) M all-trans and 13-cis RA and in ovalbumin (OVA)-sensitized BALB/c mice treated with RA (20 mg/kg) before and during sensitization. IgE and IgG1 levels were determined in the sera of the mice at day 21 after 2 injections (days 1 and 8) of aluminum hydroxide-absorbed OVA. RESULTS: All-trans and 13-cis RA inhibited in vitro IgE production from PBMC in a dose-dependent manner, but were more efficient in atopic dermatitis patients with low total serum IgE levels (< 400 kU/ml), maximal inhibition for all-trans RA at 10(-7) M (87%) and for 13-cis RA at 10(-5) M (96%) compared to patients with high serum IgE levels (>2,000 kU/ml), maximal inhibition for both all-trans and 13-cis RA at 10(-5) M (53 and 39%, respectively). In contrast, the in vivo data from OVA-sensitized mice revealed comparable total IgE and IgG1 levels in control versus all-trans RA or CD336-treated groups, specific IgE was even higher in the CD336-treated group (n = 10, 2,814 ng/ml), and was comparable in mice treated with OVA alone or with additional all-trans RA (n = 10, 1,447 and 1,354 ng/ml, respectively). CONCLUSIONS: These results indicate that the efficacy of retinoids to inhibit IgE production in vitro depends on the frequency of switched cells in the peripheral blood and that in an in vivo model using OVA-sensitized mice, retinoids fail to inhibit IgE production.     

In vitro production of IgE and IgG by sensitized human peripheral lymphocytes.

Antigenic stimulation of peripheral lymphocytes was made in two different groups of atopic patients, testing their capabilities to produce IgE and IgG in vitro. For this purpose, Marbrook's technique was used in the cultures. The immunoglobulins produced were measured in the supernatants by a sensitive double-antibody method. Results demonstrate that it is possible to produce enough IgE and IgG to be detected. Some important parameters had to be considered: appropriate selection of the patients and the antigens used in the antigenic stimulation, as well as the number of cells and the duration of the culture.     

Inhibition by cyclosporin A of IgE production and cyclophosphamide-induced eosinophilia in rats immunized with non-parasite antigens

Administration of cyclosporin A (CS-A; 25 mg/kg daily) to rats from the time of immunization with ovalbumin (OVA) in complete Freund's adjuvant abolished the production of anti-OVA antibodies, including IgE. Cyclophosphamide (Cy; 150 mg/kg) given 2 days before immunization also inhibited specific antibody production but at the same time induced a striking eosinophilia. Combined administration of both drugs resulted in the inhibition by CS-A of the Cy-induced eosinophilia. The results suggest that IgE synthesis and eosinophil proliferation may be under the control of separate T cell subsets. This rat model may prove useful in studies on the regulation of eosinophil production and the role of these cells in disease processes.     

Inhibition of T helper 2-type responses, IgE production and eosinophilia by synthetic lipopeptides

In allergy and asthma, the fine balance between the T helper (Th) 1, Th2 and T regulatory cytokine responses appears to be shifted towards Th2. Here, we report that synthetic lipopeptides which contain the typical lipid part of the lipoprotein of gram-negative bacteria stimulate a distinct regulatory cytokine pattern and inhibit several Th2 cell-related phenomena. The most potent analogue of synthetic lipopeptides, lipopeptide CGP 40774 (LP40) was not active in MyD88-deficient mice and stimulated Toll-like receptor (TLR)-2, but not TLR-4. LP40 potentiated the production of IFN-gamma and IL-10, but not IL-4 and IL-5 by human T cells. In addition, triggering of TLR-2 by lipopeptides promoted the in vitro differentiation of naive T cells towards IL-10- and IFN-gamma-producing T cells and suppressed IL-4 production by Th2 cells. Accordingly, LP40 inhibited IgE production induced by allergen, anti-IgD antibody, Nippostrongylus brasiliensis or murine acquired immunodeficiency virus. Furthermore, ovalbumin-induced lung eosinophilic inflammation was abolished and Schistosoma mansoni egg-induced granuloma size and eosinophil counts were suppressed in mice by LP40. These results demonstrate that stimulation of TLR-2 by lipopeptides represents a novel way for possible treatment of allergy and asthma by regulating the disrupted cytokine balance.     

Inhibition of herpes simplex virus production in vitro by Cyclosporin A

Summary Nontoxic concentrations of Cyclosporin A (CyA) dose-dependently inhibited herpes simplex virus (HSV) production in resting monkey kidney cells. The block was at the step of virus DNA synthesis as assessed by [³H]thymidine incorporation and by dot blot hybridization of infected cell DNA using a cloned³²P-labelled HSV DNA fragment (BamHI X) as probe. This was further supported by analysis of HSV protein synthesis in the presence of CyA as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. A relative accumulation of HSV - (e.g., ICP 4) and ₁-proteins (e.g., ICP 6 and 8) was found, whereas HSV ₁-proteins were slightly decreased and ₂-proteins were markedly decreased by CyA. The production of thymidine kinase and DNA polymerase was decreased when CyA was added to HSV infected cells. The sensitivity to CyA was not escaped by thymidine kinase nor DNA polymerase deficient mutants. Passage of HSV in presence of CyA did not result in induction of drug resistance.     

In vitro production of IgE by human peripheral blood mononuclear cells. II. Cells involved in the spontaneous IgE production in atopic patients

Spontaneous IgE production in vitro was investigated in 7-day cultures of unfractionated mononuclear cells (MNC) and MNC subpopulations from atopic patients. Depletion of either phagocytic or adherent cells decreased the amount of IgE detectable in 7-day culture supernatants, but this decrease was due, at least in part, to a loss of cytophilic IgE. Depletion of immunoglobulin-bearing cells (SIg⁺) reduced significantly but did not abolish the spontaneous IgE production in vitro. On the other hand, depletion of IgM-bearing lymphocytes (SIgM⁺), which virtually abolished the production of immunoglobulins of the IgM class, did not change significantly the spontaneous production of IgE. Similarly, no change in the spontaneous production of IgE was found when lymphocyte suspensions were depleted of complement receptor-bearing cells (CR⁺). In contrast, spontaneous IgE production was significantly increased by depletion of T lymphocytes and this increase did not simply reflect the enrichment for IgE-producing cells caused by the fractionation procedure. No significant change in the spontaneous IgE production was found when small numbers of autologous T lymphocytes were added to B cell fractions, whereas the addition of higher concentrations of autologous T cells induced a marked inhibition of the spontaneous IgE production. On the other hand, the addition in culture of pokeweed mitogen (PWM) resulted in a marked reduction of the spontaneous IgE production by B cells, also in the presence of small concentrations of autologous T lymphocytes. Normal T cells were consistently effective in inducing a partial inhibition of the spontaneous IgE production by B cells from atopic patients, whereas T cells from a noticeable proportion of atopic patients were not. These data suggest that MNC responsible for the spontaneous IgE production in atopic subjects are SIgM- and CR-deficient well-differentiated lymphocytes which probably represent the result of an activation which has occurred in vivo. However, this spontaneous IgE production can still be influenced by in vitro manipulation, such as variations in T–B cell ratios or addition of PWM. The results here reported also indicate that normal T cells are generally more effective than T cells from atopic patients in regulating the activity of spontaneous IgE-producing cells present in the blood of atopic subjects.     

Varying effects of intravenous immunoglobulin on mononuclear cell proliferation in vitro

Intravenous immunoglobulin (IVIG) is being increasingly used to treat numerous immune-mediated diseases. However, there is a paucity of knowledge on the specific mode of action of IVIG in vivo. In this study, the in vitro effects of IVIG on peripheral blood mononuclear cell (PBMC) proliferation using phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (MAb), phorbol myristate acetate (PMA), or purified protein derivatives (PPD) have been analyzed. The PBMCs were obtained from more than 10 individual donors. In all cases, IVIG almost completely inhibited PBMC proliferation at concentration above 20 mg/mL except when used in conjunction with PMA. PHA-induced proliferation of PBMCs at concentrations ranging from 1 to 15 mg/mL did not show significant differences. Anti-CD3 MAb-induced proliferation showed dose-dependent inhibition at concentrations ranging from 1 to 10 mg/mL. Interestingly, PMA-induced proliferation of PBMCs showed a dose-dependent increase at the same concentration range. PPD-induced proliferation of PBMC at concentrations ranging from 1 to 10 mg/mL did not show any statistically significant differences. These results suggest that high dose IVIG may be necessary to immune modulation in vivo and IVIG has various effects on PBMCs proliferation in limited concentration in vitro.     

Inhibition of macrophage nitric oxide production by tetrahydrocannabinol in vivo and in vitro

Δ⁹-Tetrahydrocannabinol (THC, 10 gg) was administered intraperitoneally to thioglycollate-treated mice. After 18 h, peritoneal macrophages were harvested and nitric oxide (NO·) production was induced by lipopolysaccharide (LPS, 1 μg/ml) and interferon-γ (IFN-γ, 0.1–10 U/ml). Macrophages from THC-treated mice produced about half as much NO· as controls. THC (1 μg/ml) added in vitro caused further inhibition. Greater inhibition was observed at the lower (0.1-0.3 U/ml) IFN-γ concentrations. The results suggest that the use of THC can reduce NO· production and thereby affect host defense mechanisms, inflammation and autoimmune responses.     

In vitro production of IgE by human peripheral blood mononuclear cells. I. Rate of IgE biosynthesis

IgE protein and grass-specific IgE antibodies were detected in the supernatants of 7-day cultures of unstimulated and pokeweed mitogen (PWM) stimulated human blood mononuclear cells from non-atopic and grass pollen-sensitive individuals. Significant amounts of IgE protein were detected in culture supernatants of grass-sensitive individuals and, even at lower levels, in those of non-atopic subjects. In contrast, detectable amounts of grass-specific antibodies were found only in the culture supernatants of grass-sensitive subjects. The mean values of total and grass-specific IgE detected in the supernatants of unstimulated and PWM-stimulated cultures did not differ statistically. Time sequence studies showed that IgE concentrations, measured in the 7-day supernatants, were due to a continuous release from the cells of IgE quantities progressively decreasing up to days 7 or 8. Comparison of the IgE protein and IgE antibody found in the 7-day culture supernatants to those released from initial cell pellets by treatment with acid buffer or freezing and thawing, showed that the IgE detected in 7-day supernatants could result, in part, from the release of cytophilic IgE bound to basophil or other cell types and in part also from the release of preformed lymphocyte cytoplasmic IgE into the supernatant fluids during the course of culture. In most non-atopic subjects and in some grass-sensitive patients the preformed IgE accounted virtually for the total IgE detected in the 7-day culture supernatants. However, the increase of IgE above the levels measured in the initial cell pellets, which was found in most grass-sensitive subjects, clearly reflected newly synthesized IgE. Both cycloheximide and puromycin were capable of reducing significantly the IgE concentration in culture supernatants when it was greater than the amount found in the initial cell pellets. The treatment of cells with mitomycin C was also able to decrease significantly the amount of IgE released in the supernatant after day 3 of culture.