Immunomodulation by intravenous immunoglobulin : the effect on cytokine production studied at the single-cell level

Abstract: Preparations of pooled human immunoglobulin for intravenous use (IVIg) have an established therapeutic role in immunodeficiencies, Idiopathic Thrombocytopenic Purpura and Kawasaki's disease and has been attempted in other immunologically mediated conditions including autoimmuntiy, malignancies, bone marrow transplantation and serious infections such as group A streptococcal toxic shock syndrome. The effects of IVIg on cytokine synthesis in peripheral blood mononuclear cells (PBMC) was studied in vitro by applying a new immunohistochemical method for detection of cytokine production at a singlecell level. Identification of true producer cells was possible by the accumulation of the protein in the Golgi stacks rendering the cells a characteristic staining pattern. Following superantigen stimulation, with Streptococcal pyrogenic exotoxin-A (SPE-A), co-culture with IVIg induced an almost complete block of synthesis of IFN- and TNF-ß in combination with a marked reduction in blast transformation. A significant reduction in IFN-[gamma] and TNF-ß synthesis was evident also when IVIg was added 24 hrs after activation and this in combination with a maintained monokine production argues for additional effector mechanisms in conjunction with toxin-neutralization. A novel technique based on computerized image analysis (IA) was developed to assess frequencies of producer cells, cell size and intensity of protein staining. Cells stimulated with SPE-A and co-cultured with IVIg were stained for IFN-[gamma] and evaluated. A strong correlation between manual microscopy and computerized IA was demonstrated. This study confirmed our previous findings that IVIg led to a reduced blast transformation, frequency of lymphokine producing cells with a less intense staining. PBMC were polyclonally stimulated with the protein kinase activator phorbol 12-myristate 13-acetate (PMA) in combination with the calcium ionophore ionomycin (1) which resulted in a substantial production of IL-2, IL-3, IL-4, IL- 5, IL-10, IFN-[gamma], TNF-ß and GM-CSF that was transiently suppressed during the initial 48 hrs by IVIg supplementation whereas TNF-[alpha] was unaffected. With immobilized anti-CD3 mAb activation IVIg led in a sustained reduction of synthesis of IL-2, IL-10, IFN-[gamma], TNF-ß and TNF-[alpha] throughout the 96 hrs of culture. The monokine IL-8 was completely unaffected. IVIg also induced a decreased proliferation and blast transformation and for anti-CD3 stimulated cells a reduced IL-2 receptor expression was also evident. To evaluate whether IVIg targeted T cells, directly or via accessory cells, T cells were purified from PBMC and activated polyclonally with PMA/I or immobilized anti-CD3 mAb and co-cultured with IVIg in parallel with cultures containing unfractionated PBMC. An inhibition of synthesis of IL-2, IFN-[gamma], TNF-ß and GM-CSF was seen in PBMC whereas the production in purified T cells was unaffected or even increased. This indicated that the inhibitory effect on T cell activation by IVIg was mediated via accessory cells. The influence on Th2 type lymphokines was assessed in PBMC activated with bacillus CalmetteGuérin (BCG) for 12 days. An initial Th1 type response, dominated by IFN-[gamma] and TNF-ß, was followed by Th2 pattern with production of IL- 4, IL-5 and IL-10. Addition of IVIg resulted in a general down-regulation or both types of lymphokines suggesting an inhibitory effect irrespective of the Thl/Th2 balance. In conclusion, this study shows that IVIg has a strong inhibitory influence ex vivo on cytokine synthesis. A decrease in proliferation, blast transformation and lymphokine synthesis was observed. These potent immunomodulatory properties by IVIg may in part explain the beneficial influence on inflammatory diseases such as Kawasaki's vasculitis. Furthermore, these results support clinical trials with IVIg therapy in septic shock syndrome caused by superantigen-producing gram-positive bacteria.