Benzylpenicillin differentially conjugates to IFN-gamma, TNF-alpha, IL-1beta, IL-4 and IL-13 but selectively reduces IFN-gamma activity

Abstract

It is known that beta-lactam antibiotics can conjugate to lysine and histidine residues on proteins via the carbonyl group of the opened beta-lactam ring. However, it is not known which proteins these drugs target and there is little work addressing whether conjugation is preferential for some proteins over others or if conjugation has functional consequences for the protein. We have previously shown that the beta-lactam antibiotic benzylpenicillin (BP) conjugates to IFN-gamma and reduces its activity. This interaction demonstrates selectivity, as BP does not bind to IL-4. Here, we extend our study to include other Th1 and Th2 cell-associated cytokines and two cytokines associated with inflammatory responses. We demonstrate by Western blotting that BP also conjugates to IL-1beta, IL-2, IL-5, IL-13 and TNF-alpha but not to IL-10. Densitometric analysis of leading cytokine bands on blots revealed that IFN-gamma always gave more intense BP-positive bands than any other cytokine analysed. Cytokines pre-incubated with BP at 37 degrees C in a protein-containing, serum-free medium were assayed for their biological activity. By in vitro bioassay, BP inhibited the ability of IFN-gamma but not IL-1beta or TNF-alpha to induce CD54 expression on epithelial cells. In addition, BP did not affect IL-4 or IL-13 inhibition of mast cell proliferation. When the pre-incubation temperature was reduced to 4 degrees C, BP did not conjugate to IFN-gamma or modulate its activity. BP retained its inhibitory effect on IFN-gamma activity when 20% FCS was added to the pre-incubation medium. In conclusion, BP conjugates to some cytokines but not others and this does not appear to be related to primary protein structure. Furthermore, of the cytokines studied, conjugation only to IFN-gamma is accompanied by inhibition of activity. This phenomenon is temperature dependent and occurs in the presence of serum. These findings provide further evidence for differential, direct drug-cytokine interactions. Such interactions may have therapeutic implications in terms of targeting cytokines to regulate their activity.

Figures

Fig. 1

BP conjugation to cytokines. Cytokines, each at 10 µg/ml in PBS, were incubated with or without 5 mg/ml BP overnight at 37°C. Samples were analysed by SDS-PAGE and Western blotted with rabbit polyclonal anti-BP antibody. In each case, one blot representative of three is shown. m, murine.

Fig. 2

BP has no effect on cytokine oligomerization. IFN-γ, IL-2 and TNF-α, each at 10 µg/ml in PBS, were incubated with or without 5 mg/ml BP overnight at 37°C. Samples were analysed by SDS-PAGE and Western blotted with the appropriate polyclonal anti-cytokine antibody.

Fig. 3

Effect of BP on the ability of(a)IL-4 or(b)IL-13 to inhibit HMC-1 cell proliferation. Each cytokine (100 ng/ml) was incubated in RT2 with or without BP (2 mg/ml) for 4 days at 37°C, then each preparation was assayed at 2, 10 or 50 ng/ml. Proliferation was measured as uptake of 3H-thymidine after cells had been incubated for 4–6 days with the IL-4 or IL-13 preparations. Results represent mean ± SEM of 4 experiments, each performed in replicates of at least 4. *P < 0·05, by paired Student's t-test, for comparison of cells cultured with or without cytokine. ▪ No cytokine, □ Cytokine incubated 4 days alone, Cytokine incubated 4 days + 2 mg/ml BP, Cytokine incubated 4 days alone, 2mg/ml BP added immediately before assay.

Fig. 4

Effect of BP on the ability of(a)IL-1β,(b)TNF-α or(c)IFN-γ to induce CD54 expression on A549 cells. Each cytokine (100 ng/ml) was incubated in RT2 with (○) or without (•) BP (2 mg/ml) for 4 days at 37°C, then each preparation was assayed at 0·02, 0·2, 2 or 10 ng/ml. Confluent layers of A549 cells in 96-well plates were incubated overnight with cytokine preparations. After fixing, CD54 expression was measured by ELISA. ▴ Cytokine incubated 4 days alone, 2 mg/ml BP added immediately before assay. Results represent mean ± SEM of 3 (IL-1β and TNF-α) or 4 (IFN-γ) experiments, each performed in triplictate. *P < 0·05, by paired Student's t-test, for comparison of untreated to BP-treated IFN-γ.

Fig. 5

Effect of temperature on BP conjugation to IFN-γ. IFN-γ, at 10 µg/ml in PBS, was incubated with or without 5 mg/ml BP overnight at 4 or 37°C. Samples were analysed by SDS-PAGE and Western blotted with rabbit polyclonal anti-BP antibody.

Fig. 6

Effect of temperature and serum on BP reduction of IFN-γ activity. IFN-γ was incubated, at 100 ng/ml, at different temperatures in RT2, or in varying concentrations of FCS for 4 days with or without 2 mg/ml BP. Confluent layers of A549 cells in 96-well plates were incubated overnight with each cytokine preparation at a final concentration of 2 ng/ml. After fixing, CD54 expression was measured by ELISA. Results represent mean ± SEM of 4 experiments, each performed in triplictate.*P < 0·01 by paired Student's t-test, for comparison of untreated to BP-treated IFN-γ.□ medium alone, IFN-γ, ▪ IFN-γ+ BP.