In vitro interleukin 4 and interferon-gamma production by mononuclear cells from atopic dermatitis patients

In order to elucidate further the possible role of specific cytokines in the pathogenesis of atopic dermatitis (AD) the in vitro production of interleukin 4 (IL-4) and interferon-gamma (IFN-γ) in patients with severe atopic dermatitis (n = 4) was compared with that in a group of non-atopic healthy controls. Overall IL-4 production by PHA- and PWM-driven PBMNCs was increased in controls during the first 48 h in culture. Addition of interleukin 2 (IL-2) into parallel cultures generated an insignificant (p > 0.05) increase in IL-4 production in AD patients compared with that from controls. IFN-γ production by PWM-stimulated PBMNCs was markedly decreased in AD patients compared with controls (p < 0.01). Addition of IL-2 (250 U/ml) to parallel cultures failed to restore IFN-γ production in AD patients. Finally, no IL-4 or IFN-γ activity could be detected in any of the sera. In conclusion, the data suggest a possible dysregulation of cytokine production in at least a subgroup of AD patients, with an impaired capacity to secrete IFN-γ, but a partially intact IL-4 generating capacity.


Introduction
Although atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disorder first described as a disease entity in the early decades of this century, its etiopathogenesis has not yet been fully elucidated. In addition to genetic disposition, AD is influenced by many environmental factors. Numerous humoral and cellular immunological abnormalities have been reported in AD, including high IgE levels, increased numbers of IgE-Fc receptor (FceR) bearing lymphocytes, and cellular dysfunctions in T-cells. In the murine system 2'3 as well as in humans 4 T-helper cells can be divided into two subsets (TH1 and TH2) on the basis of the cytokines they produce: TH cells secrete interleukin 2 (IL-2) and interferon-gamma (IFN-2) and TH2 cells secrete interleukin 4 (IL-4) and interleukin 5 (IL-5). Evidence has accumulated that the human IgE response is mainly regulated by the relative production of the reciprocally active cytokines IL-4 (TH2 system) and IFN-2 (TH system), s-8 Both recombinant and natural IL-4 induce IgE synthesis by peripheral blood mononuclear cells (PBMNCs) from atopic and healthy donors. 9-IFN-y downregulates this IL-4 induction of IgE synthesis in vitro, s'2-14 It has been suggested that in atopic individuals allergens may 'select' T-helper cells with peculiar profiles of cytokine production (high IL-4 and low IFN-,), thus contributing to the pathogenesis of IgE-mediated disorders. 6 Only a few studies have investigated the simultaneous production of IL-4 and IFN-y in vitro using activated PBMNCs cultures from atopic donors. s- 7 The results have been in part controversial with respect to IL-4 s-7'18 and IFN-/16'7'9'2 production. These data prompted the authors to examine whether IL-4 and IFN-2 activity could be detected in the supernatants (SN) of plant lectin-stimulated (PHA and PWM) PBMNC cultures from AD patients with moderate to severe disease activity using commercially available ELISAs. 2 In order to investigate the in vitro effect of IL-2, a T-cell growth factor described as being 22   did not dier significantly from controls (data not shown).
EJfects of IL-2 supplementation. In order to investigate the IL-2 requirement for IL-4 generation, rhlL-2 (250 U/ml) was added with PWM or PHA. IL-2 supplementation did not increase IL-4 production significantly in AD patients and controls. We further investigated whether IL-2 can restore the impaired IFN-2 production in AD patients. No significant difference was observed between IFN-2 secretion (at 24 h) upon PWM plus IL-2 stimulation compared with PWM stimulation alone (Fig. 3) or PHA plus IL-2 stimulation compared with PHA stimulation alone.
Addition of IL-2 (250 U/ml) to PWM-stimulated PBMNCs (black bars) of AD patients did not significantly increase IL-4 production (at 24 27 where serum concentrations of IL-4 could be measured in children with allergic diseases as well as in non-allergic controls. Differences in the IL-4 assays used might partly account for these discrepancies. More importantly, in vitro PWM-induced IFN-y production by PBMNCs from AD patients was significantly lower compared with controls ( Fig. 2).
An accepted view is that the elevated serum IgE levels in atopic individuals are a consequence of imbalances between IL-4-producing and IFN-2producing helper T-cells. 28 Parronchi et al. 29 suggested that the high amounts of IL-4 without simultaneous IFN-y production by allergen-specific TH cells induces the formation of IgE antibodies.
The defective IFN-2 production in AD patients observed here might not be able to successfully down-regulate IL-4-induced IgE synthesis in vivo in these individuals, whereas high IFN-2 levels in healthy controls might successfully antagonize the The observed positive correlation in AD patients between in vitro IFN-y production and total serum IgE is in apparent conflict with the view that IFN-y down-regulates IgE production and in contrast with another report. 31 The data presented here do not demonstrate a significant difference between PHA/PWM-induced IL-4 production in AD patients and controls (Fig.  2). Supplementation with IL-2 (250 U/ml) failed to induce a significant increase in IL-4 generation from PHA/PWM-stimulated PBMNCs from AD patients. Seder et al. 35 have shown that the frequency of IL-4 producing cells is very low among circulating antigen-primed T-cells; furthermore several groups recently reported evidence for the accumulation and expansion of T-lymphocytes producing high amounts of IL-4 in lesional skin, whereas T-lymphocytes from peripheral blood were poor IL-4 producers. 36'37 Further investigations should compare the cytokine profiles from lesional skin cells and the data obtained here from peripheral blood cells in the same AD individuals. However it is remarkable that, despite the now accepted view that IFN-), and IL-4 predominantly act at a local level, a highly significant decreased in vitro IFN-2 production can be observed among peripheral blood MNCs.
In contrast to IL-4, no correlation between IFN-2 production and cell proliferation was observed.
However, Patarca et al.38 demonstrated that activation of TH1 cells does not necessarily result in IFN-y production.
In conclusion, although the number of investigated individuals is small, we could demonstrate a defective in vitro IFN-y production in PBMNCs from patients with mild to severe forms of AD and moderate to high total serum IgE concentrations, whereas no significant difference in IL-4 secretion was observed in these patients when compared with controls.