Alpha-1-antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in whole blood

Abstract

Several observations suggest endogenous suppressors of inflammatory mediators are present in human blood. alpha-1-Antitrypsin (AAT) is the most abundant serine protease inhibitor in blood, and AAT possesses anti-inflammatory activity in vitro and in vivo. Here, we show that in vitro stimulation of whole blood from persons with a genetic AAT deficiency resulted in enhanced cytokine production compared with blood from healthy subjects. Using whole blood from healthy subjects, dilution of blood with RPMI tissue-culture medium, followed by incubation for 18 h, increased spontaneous production of IL-8, TNF-alpha, IL-1 beta, and IL-1R antagonist (IL-1Ra) significantly, compared with undiluted blood. Dilution-induced cytokine production suggested the presence of one or more circulating inhibitors of cytokine synthesis present in blood. Serially diluting blood with tissue-culture medium in the presence of cytokine stimulation with heat-killed Staphylococcus epidermidis (S. epi) resulted in 1.2- to 55-fold increases in cytokine production compared with S. epi stimulation alone. Diluting blood with autologous plasma did not increase the production of IL-8, TNF-alpha, IL-1 beta, or IL-1Ra, suggesting that the endogenous, inhibitory activity of blood resided in plasma. In whole blood, diluted and stimulated with S. epi, exogenous AAT inhibited IL-8, IL-6, TNF-alpha, and IL-1 beta significantly but did not suppress induction of the anti-inflammatory cytokines IL-1Ra and IL-10. These ex vivo and in vitro observations suggest that endogenous AAT in blood contributes to the suppression of proinflammatory cytokine synthesis.

Fig. 1.

Effect of AAT deficiency on cytokine production in stimulated whole blood. Cytokine production was measured in undiluted, S. epi-stimulated blood obtained from nine AAT-deficient patients (•) and from 10 healthy controls (▪). After 18 h of stimulation, supernatants were removed for cytokine assays, including IL-8 (A), IL-6 (B), TNF-α (C), IL-1β (D), IL-1Ra (E), and IL-10 (F). Horizontal bars indicate median levels. **, P < 0.005, and ***, P < 0.0005, compared with healthy controls.

Fig. 2.

Spontaneous cytokine production in whole blood diluted with RPMI. Heparinized whole blood was incubated for 18 h undiluted (dilution=0) or diluted in RPMI to the final concentrations indicated on the horizontal axes. After incubation, supernatants were removed, and cytokine levels were quantified. Cytokine levels are depicted as measured concentrations (A, C, E, and G) or multiplied by the dilution factors (B, D, F, and H). Shown are concentrations of IL-8 (A, B), TNF-α (C, D), IL-1β (E, F), and IL-1Ra (G, H). Cytokine concentrations are indicated on the vertical axes as means ± sem in four separate donors. *, P < 0.05; **, P < 0.01, compared with dilution = 0.

Fig. 3.

Effect of whole blood dilution on S. epi-stimulated cytokine production. Heparinized whole blood was incubated for 18 h undiluted (dilution=0, far-left bars), incubated undiluted with heat-killed S. epi as a stimulus (dilution=0, second bars from left), or with RPMI dilution to the levels indicated on the horizontal axes and with S. epi stimulation. After incubation, cytokine concentrations were measured and expressed as concentration per mL of blood (multiplied by dilution factor) for IL-8 (A), IL-6 (B), TNF-α (C), IL-1β (D), IL-1Ra (E), and IL-10 (F). Cytokine concentrations are indicated on the vertical axes as means ± sem in four separate donors. *, P < 0.05, and **, P < 0.01, compared with cultures stimulated with S. epi in the absence of dilution (dilution=0, second bars from left).

Fig. 4.

Cytokine production in whole blood diluted with RPMI or autologous plasma. Whole blood was incubated for 18 h undiluted (dilution=0), diluted 1:32 in RPMI [1:32 (RPMI)], or diluted 1:32 in autologous plasma [1:32 (plasma)]. Supernatant cytokine concentrations are reported without multiplication by the dilution factor. Shown are levels of IL-8 (A), TNF-α (B), IL-1β (C), and IL-1Ra (D). Cytokine concentrations are indicated on the vertical axes as means ± sem in eight separate donors. For IL-8, **, P < 0.01, compared with dilution = 0; for TNF-α, *, P < 0.05, compared with dilution = 0; for IL-1β, *, P < 0.05, compared with dilution = 0 and to 1:32 (plasma); and for IL-1Ra, **, P < 0.01, compared with 1:32 (plasma).

Fig. 5.

Effect of exogenous AAT on cytokine production in diluted whole blood with S. epi stimulation. Whole blood was diluted 1:32 in RPMI and stimulated with heat-killed S. epi in the absence (AAT=0) or presence of AAT added 1 h prior to S. epi. Final AAT concentrations are shown on the horizontal axes. Supernatant cytokine concentrations were measured and expressed as concentrations per mL of blood (multiplied by dilution factor). Shown are levels of IL-8 (A), IL-6 (B), TNF-α (C), IL-1β (D), IL-1Ra (E), and IL-10 (F). Cytokine concentrations are shown on the vertical axes as means ± sem in cultures from four separate donors. **, P < 0.01, compared with AAT = 0.

Fig. 6.

Effect of the synthetic serine Pi AAPV-CMK on cytokine production in diluted and S. epi- stimulated whole blood, which was diluted 1:16 in RPMI (Control), diluted 1:16 in RPMI and stimulated with S. epi (S.epi), or diluted 1:16 in RPMI and stimulated with S. epi in the presence of 50 μM AAPV-CMK, added 1 h prior to S. epi (S.epi+AAPV-CMK). After 18 h of incubation, supernatant cytokine concentrations were determined and expressed as concentration per mL of blood (multiplied by dilution factor). Shown are levels of IL-8 (A), IL-6 (B), TNF-α (C), IL-1β (D), and IL-1Ra (E). Cytokine concentrations are shown on the vertical axes as means ± sem in four separate donors. *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with S. epi.