Etienne Dupont

In vitro inhibition of tumour necrosis factor-α and interleukin-6 production by intravenous immunoglobulins

In vitro data about the action of pooled immunoglobulins (Ig) on cytokine (CK) production are controversial. The recent finding of natural antibodies against staphylococcal toxins neutralizing superantigen‐induced activation prompted us to design an assay determining their ability to modulate staphylococcal enterotoxin B (SEB) induced CK production (IL‐6 and TNF‐α). Presence of anti‐SEB antibodies was demonstrated by a dot‐blot assay in the three preparations tested. Preincubation of SEB with pooled Ig prior to addition into the test tube containing PBMCs (neutralizing condition) resulted in a strong inhibition of both TNF‐α and IL‐6 release (TNFα: 59 ± 5% inhibition, P<0.0001; IL‐6: 71 ± 7% inhibition. P<0.0001, n= 15). Anti‐CD3 MoAb‐induced CK production was not modified. During our study it was found that experimental conditions were critical to observe this inhibitory effect. Reversing the previous procedure by adding PBMCs into the test tube containing pooled Ig mixed with SEB resulted in a marked induction of TNF‐α and IL‐6 production. The same observation was made when pooled Ig solely was added (coating condition). F(ab)2 fragments of pooled Ig displayed similar inhibitory capacity when added in neutralizing condition, indicating that the mechanism involved was not Fc dependent. The fragments lost the activating properties of intact Ig when incubated in coating condition, showing that Fc receptor activation occurs in this setting.

Blockade of proliferation and tumor necrosis factor-alpha production occurring during mixed lymphocyte reaction by interferon-gamma-specific natural antibodies contained in intravenous immunoglobulins.

The mechanism of action of intravenous immunoglobulins (IVIg) for prevention of graft rejection and graft-versus-host disease (GVHD) is poorly understood. Recently, it has been shown that these preparations contain natural antibodies directed toward interferon (IFN)-gamma. During mixed lymphocyte reaction (MLR), which constitutes an in vitro model of allograft rejection and GVHD, T cell recognition of HLA differences induces IFN-gamma release. This cytokine promotes T cell proliferation and acts as a macrophage-activating factor to provoke tumor necrosis factor-alpha secretion. The aim of the present work is to investigate the influence of IVIg on IFN-gamma production occurring during MLR and its subsequent impact on T cell proliferation and tumor necrosis factor (TNF)-alpha secretion. We tested IVIg preparations for the presence of anti-IFN-gamma and anti-TNF-alpha antibodies. High amounts of anti-IFN-gamma, but not anti-TNF-alpha antibodies, were found. IVIg addition at the initiation of culture resulted in IFN-gamma secretion blockade. Likewise, lymphocyte proliferation and TNF-alpha secretion were inhibited. This inhibition was reversed by the addition of recombinant human IFN-gamma. Furthermore, the inhibitory properties of IVIg were mimicked by an IFN-gamma-specific neutralizing monoclonal antibody. We conclude that the capacity of IVIg to inhibit proliferation and TNF-alpha release during MLR is due to IFN-gamma blockade by natural antibodies. This immunosuppressive mechanism could contribute to the effect of IVIg on prophylaxis of organ graft rejection and GVHD after allogeneic bone marrow transplantation.

Optimal control of interferon-gamma and tumor necrosis factor-alpha by interleukin-10 produced in response to one HLA-DR mismatch during the primary mixed lymphocyte reaction.

Interleukin (IL)-10 is an immunosuppressive cytokine potentially involved in the control of the allogeneic response. Several studies failed to detect it in mixed lymphocyte reaction supernatants. However, experiments using IL-10-specific antibodies, revealing its inhibitory action on interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, provided indirect evidence that endogenous IL-10 was produced. The aim of the present work is to elucidate the role of IL-10 during mixed lymphocyte reaction and to investigate the influence of HLA-DR antigens on its production and on the regulatory loop involving TNF-alpha and IFN-gamma. Using a highly sensitive ELISA, a significant (P < 0.0001) but low IL-10 release could be detected (33.7 +/- 3.6 pg/ml) in response to HLA-DR disparities. However, IL-10 release was not graded as 1 DR mismatch (MM)-induced maximal secretion (32.3 +/- 5.1 pg/ml). This contrasted with TNF-alpha and IFN-gamma productions, which significantly increased in 2 DR MM pairs. Addition to IL-10-specific antibodies resulted in higher enhancement of INF-gamma (235 +/- 38% vs. 122 +/- 39%, P = 0.02) and, to a lesser extent, TNF-alpha (147 +/- 56% vs. 112 +/- 20%, NS) in 1 compared with 2 DR MM pairs. We conclude that the 1 DR MM setting is associated with optimal IL-10 secretion and more efficient inhibition of IFN-gamma and TNF-alpha compared with the 2 DR MM configuration. Although promoting enhanced IFN-gamma and TNF-alpha release, introduction of an additional DR MM does not result in increased IL-10 production. These data indicating that the IL-10 regulatory feedback loop is more effective in 1 DR rather than complete DR incompatibility could have an impact on matching policies for planned transfusion.

Increased in vitro Immunosuppressive Action of Anti-CMV and Anti-HBs Intravenous Immunoglobulins due to Higher Amounts of Interferon-Gamma Specific Neutralizing Antibodies

Background and objectives: We previously found that interferon‐γ (IFN‐γ) antibodies in intravenous immunoglobulins (IVIG) can block not only IFN‐γ production and tumor necrosis factor‐α secretion, but also T‐cell proliferation. Since the presence of IFN‐γ antibodies has been attributed to previous viral infection, we hypothesized that the viral status of the plasma donors used for IVIG pools might be a decisive factor in controlling the immunosuppressive capacity of IVIG. Materials and methods: We tested three different pooled, human IVIG preparations for the presence of IFN‐γ antibodies by ELISA. Results: Comparison of the immunomodulatory activity of polyvalent IVIG with that of specific CMV and HBs IVIG showed that the latter had higher levels of IFN‐γ antibodies and an increased capacity to block mixed lymphocyte reaction and cytokine production. Conclusion: We propose that these in vitro assays constitute a basis for the selection of plasma intended for manufacturing IVIG aimed at immunosuppression in the transplant setting.

INTERLEUKIN 12 UNMASKS HLA CLASS I DIFFERENCES DURING MIXED LYMPHOCYTE REACTION INDUCED INTERFERON GAMMA PRODUCTION

We investigated the genetic control of IFN-gamma release during MLR and its relationship with TNF-alpha and IL-12. Blocking experiments demonstrated the IFN-gamma dependence of TNF-alpha production and the significant contribution of IL-12 to IFN-gamma secretion. We studied informative pairs allowing the evaluation of the relative importance of HLA class I and class II antigens. Maximal IFN-gamma secretion allowing discrimination between fully HLA different and identical subjects required 5 days. In class I different but DRB1 identical pairs, a moderate but discriminant IFN-gamma release was found. Exogenous IL-12 addition after 24 hours of preactivation by MLR resulted in a marked enhancement of IFN-gamma production at day 2. In pairs differing only by class I antigens, the discriminating capacity was significantly increased as compared to values obtained in absence of IL-12 at day 2 (p < 0.004) and at day 5 (p < 0.004). The crucial role of class I antigens on IFN-gamma release was further substantiated by the blocking action of the W6/32 mAb directed against a monomorphic epitope common to all HLA-A, -B, and -C antigens. We conclude that IFN-gamma production during MLR is under the control of class I antigens. Furthermore, exogenous IL-12 strongly amplifies their influence.