Reply to wadhwa et al

Abstract

We thank Drs Wadhwa, Barrowcliffe and Thorpe for their comments regarding our paper (Hodge et al, 1999). The authors comment that our study complements the results of previously published studies in which bioassays were used to measure the inhibitory effect of plasma-derived concentrates on interleukin (IL)-2 production (Thorpe et al, 1989; Wadhwa et al, 1992, 1994, 1995). These studies used bioassays to measure cytokine levels in isolated cell culture systems and have provided an important background to the data and proposals. Our study goes significantly further than these previous studies in that it documents the modulatory effect of plasma-derived factor VIII on the synthesis of many cytokines for a variety of minor leucocyte subtypes. As well as inhibition of IL-2 synthesis, both the percentage and MFI (reflecting quantity produced per cell) for T cells producing tumour necrosis factor α (TNF-α) and interferon γ (IFN-γ) production were inhibited in the presence of plasma-derived factor concentrate. For all T-cell cytokines, the inhibition was partially corrected for by addition of anti-TGF-β. Wadhwa et al also pointed out that, on addition of plasma-derived factor concentrate, we also observed limited inhibition of TNF-α, IL-1α, IL-1β, IL-6 and IL-8 by lipopolysaccharide (LPS)-stimulated monocytes and of IFN-γ production by phytohaemagglutinin (PHA) + IL-12 stimulated natural killer (NK) cells. The inhibition of these cytokines could not be corrected for by the addition of neutralizing TGF-β antibody to factor concentrate. These findings provide direct evidence that there may be other immunomodulatory agents in the factor concentrates responsible for the observed effects on monocyte and NK cell cytokine production. We also showed that the effect of plasma-derived factor concentrate on leucocyte cytokine production is immunomodulatory rather than inhibitory as the synthesis of IL-10 by LPS-stimulated monocytes was increased by the presence of plasma-derived factor concentrate. This is a very significant finding as IL-10 provides the main negative regulatory feedback mechanism in Th1-type cytokine synthesis (Romagnani, 1997). This, together with the significant decrease in all Th1-type cytokines (IL-2 by T cells, IFN-γ by T cells and NK cells and IL-12 by monocytes) noted in the presence of plasma-derived factor concentrate may explain the increase in these patient recipients of certain types of infections (e.g. mycobacterial) that require Th1 cytokine production for an effective response (Beddal et al, 1985; Mosman & Sad, 1996). In our study, whole blood culture was used, which is a more complex system than the use of bioassays to measure single cytokines produced by purified cells. Whole blood culture maintains many of the complex physiological interactions of all cell types as leucocytes produce an array of cytokines that interact with and affect each other in an immune response. The use of multiparameter flow cytometry enabled the modulatory effects of plasma-derived factor concentrates on each cytokine produced by a particular leucocyte subtype to be evaluated after this complex interaction. Wadhwa et al also correctly observed that our study only involved one fractionated concentrate (CSL Bioplasma, Parkville, Australia) and one recombinant DNA product. Immunomodulatory effects of these products were of interest to us as they were the only two factor VIII concentrates available in Australia to treat patients with haemophilia and have not been included in any previous studies. We have recently observed (unpublished observations) that the inhibitory effect of some batches of the plasma-derived factor VIII concentrate used in our study on T-cell IFN-γ secretion differed significantly. The inhibitory effect of three different batches of this fractionated concentrate on T-cell IFN-γ synthesis is shown in Fig l. Bar graph showing the inhibitory effect of three different batches of plasma-derived factor VIII concentrate (2·5 IU/ml) on T-cell IFN-γ synthesis (means ± 2SD from five experiments) compared with recombinant factor VIII (rhFVIII). All batches of plasma-derived factor concentrate significantly inhibited IFN-γ synthesis (Wilcoxin, P<0·05). The inhibitory effect of batch 2 on IFN-γ synthesis was significantly less than that of batches 1 and 3 (Wilcoxin, P<0·05). The presence of rhFVIII had no significant effect on IFN-γ synthesis. Asterisk, significant inhibition of IFN-γ production. It is therefore not valid to assume that the immunomodulatory activity shown by one batch of a particular factor concentrate will be representative of all batches of that product. We agree with Wadhwa et al that each batch of product must be evaluated on a case-by-case basis.