Figure 5. Engagement of integrin α_Dβ₂ induces expression and release of IL-8 by human monocytes.

Unfractionated human monocytes were incubated in wells coated with immobilized mAb against α_D, or in wells coated with HSA or IgG1 as control conditions, for various times. Supernatants were collected and analyzed for IL-8 by ELISA. In some experiments engagement of α_Dβ₂ was compared to engagement of other leukocyte integrins using immobilized mAb against individual leukocyte integrin α subunits. A. Engagement of α_Dβ₂ by immobilized anti-α_D mAb induced time-dependent release of IL-8, whereas IL-8 secretion by monocytes incubated on HSA- or IgG1-coated control surfaces was much lower. In parallel, incubation of monocytes with LPS (100 µg/ml) in suspension induced release of IL-8 at 8 (1.1 ng/ml) and 18 hr (4.4 ng/ml) (not shown). Engagement of αMβ₂ also induced time-dependent release of IL-8. B. Release of IL-8 triggered by engagement of α_Dβ₂ on monocytes was dependent on the concentration of anti-α_D mAb used to coat the wells. This figure indicates the results from an 8 hr incubation of monocytes on the triggering and control surfaces. C. Engagement of integrin α_Dβ₂ was a potent stimulus for IL-8 secretion at 8 hr when immobilized mAb against α_D were compared to immobilized mAb against other leukocyte integrin α subunits. Two activating anti-α_D monoclonal antibodies, 169B and 217I, were examined in this experiment. The data in Panels A-C are individual determinations in single experiments. Data from additional experiments done at the 8 hr time point using monocytes from multiple different donors are shown in Tables S3 and S4. D. Wells were coated with human albumin or recombinant ICAM-3, monocytes were incubated in these wells for 18 hr. alone, in the presence of a blocking anti-α_D mAb (mAb 240I), or in the presence of a non-blocking anti-α_D monoclonal antibody (mAb 169A). IL-8 was then measured in the supernatants. The figure indicates the results of incubations with monocytes from 8 different subjects studied in 5 separate experiments on different days. Results from each subject are identified by a different symbol. The horizontal bars in the columns of data points indicate the means of determinations for each condition. The data were analyzed with Tukey's multiple comparison test and the Neuman-Kuels multiple comparison test, with similar results in each case. The significance values from the Neuman-Kuels analysis are shown (** = p<0.001; * = p<0.01). There was no difference in release of the IL-8 when monocytes were incubated with ICAM-3 alone versus incubation with ICAM-3 in the presence of the non-blocking mAb (p>0.05).