Fig. 4

NO-mediated amplification of cytokine production is Ca ²⁺ dependent

(A) Stimulatory effect of Ca²⁺ inducers is amplified by HIV-1 and NO. HIV-1-infected and replicate uninfected monocyte cultures were treated with Ca²⁺ inducers thapsigargin (thapsi, 0.1 µM) and A23187 (2 µM). To substitute for HIV-specific NO, uninfected cultures were also treated with SNAP (100 µM). TNF levels were assayed by ELISA 18 hr after stimulation. Values are scaled relative to uninfected cultures treated with thapsigargin and A23187 alone (control, 100%). (B) Chelators of Ca²⁺ eliminate stimulatory effects of HIV-1 and NO on LPS-induced cytokine production. HIV-1-infected and uninfected (untreated [control] or treated with 100 µM SNAP) monocyte cultures were treated with 0.5 ng/ml LPS in combination with Ca²⁺ chelators EGTA (3 mM) and BAPTA (3 mM). Eighteen hours after stimulation, TNF levels were assayed in the supernatants by ELISA, and results were scaled relative to controls (100%). Error bars for Panels A and B represent SD from a representative experiment (out of two performed with cells from different donors) done in triplicate (i.e., in three independent wells), and statistical analysis was performed as in Fig. 1.