Neutrophils interact with adenovirus vectors via Fc receptors and complement receptor 1

Abstract

Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of Fc gammaRI (CD64), Fc gammaRII (CD32), and Fc gammaRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors.

FIG. 1.

Neutrophil-adenovirus interaction in vivo. (A) Total liver leukocytes at 3 h following systemic administration of AdLuc or vehicle control. Each bar is subdivided to show the differential recruitment of leukocyte subsets. For neutrophils with AdLuc versus vehicle control, P < 0.005 (***); n = 6 to 14. Error bars indicate standard deviations. (B) Flow cytometry analysis of liver leukocytes from mice 3 h following intravenous administration of Cy2-AdLuc or vehicle control. Leukocytes were analyzed alone (lower panels) or stained with PE-conjugated isotype control rat IgG2a (middle panels) or anti-Ly-6G antibody (upper panels). The fluorescence histogram shows a subpopulation of Cy2-high, Ly-6G-positive liver leukocytes in Cy2-AdLuc-treated mice. (C) Percentage of Cy2-high cells within the Ly-6G-positive neutrophil population. For AdLuc versus vehicle control, P < 0.005 (***); n = 6. (D) PCR analysis of viral DNA in Cy2-high and Cy-2-low liver leukocyte fractions. Lane 1, Cy2-high, Ad5 fiber. Lane 2, Cy2-high, GAPDH. Lane 3, Cy2-low, Ad5 fiber. Lane 4, Cy2-low, GAPDH.

FIG. 2.

Neutrophil-adenovirus interaction in vitro. (A) Neutrophils were incubated with Cy2-AdLuc vectors for 20 to 60 min and mean neutrophil fluorescence analyzed by flow cytometry. For 60 min versus cells alone, P < 0.005 (***); n = 8 to 10. Error bars indicate standard deviations. (B) Fluorescence microscopy of neutrophils incubated with Cy2-AdLuc at 60 min (Cy2, green; DAPI, blue). Magnification, ×60. (C) Electron microscopy of neutrophils incubated with AdLuc at 20 min.

FIG. 3.

Effect of neutrophils on AdLuc transduction of target cells. (A) Luciferase activity at 24 h in REC cells transduced with vehicle (−), AdLuc pretreated with fetal bovine serum (FBS), AdLuc pretreated with complete human serum (CHS), or AdLuc pretreated with complete human serum and neutrophils (CHS + neuts). For CHS versus CHS + neuts, P < 0.05 (*); n = 3. (B) Luciferase activity at 6 h following AdLuc transduction in REC cells coincubated with an increasing number of neutrophils (neutrophil/REC ratio, 25:1, 5:1, or 1:1). For no neutrophils versus coincubation ratio of 5:1 or 1:1, P < 0.05; n = 3. Error bars indicate standard deviations.

FIG. 4.

Role of capsid RGD in neutrophil-adenovirus interaction. (A) Transduction efficiency of Cy2-labeled and unlabeled AdL.PB* compared to AdLuc in REC cells. (B) Neutrophils were incubated with Cy2-AdLuc (open bars) or Cy2-AdL.PB* (closed bars) and fluorescence analyzed at 20 to 60 min. For AdLuc versus AdL.PB* at 20 min, P < 0.05 (*); n = 3. (C) Neutrophil L-selectin expression in response to Ad vectors. Neutrophils were incubated with vehicle (open bars), unlabeled AdLuc (hatched bars), or AdL.PB* (closed bars). L-selectin expression was determined by flow cytometry at 20 to 60 min For AdLuc or AdL.PB* versus vehicle, P < 0.05 (*); n = 5. Error bars indicate standard deviations.

FIG. 5.

Role of serum factors in neutrophil-adenovirus interaction. (A) Cy2-AdLuc was incubated with neutrophils and modified human serum. Cells were analyzed by flow cytometry at 20 (open bars), 40 (hatched bars), and 60 (closed bars) min. −Serum, no serum; CHS, complete human serum; −IgG, IgG-depleted human serum; HI HS, heat-inactivated human serum; −IgG HI HS, IgG-depleted heat-inactivated human serum; HI BS, heat-inactivated bovine serum. For CHS versus relevant time points from subsequent treatment groups, P < 0.01 (**); n = 3 or 4. (B) Cy2-AdLuc was incubated with neutrophils in the presence of EGTA (blocks classical complement pathway) or 50°C heat-inactivated human serum (blocks alternative complement pathway). Cells were analyzed by flow cytometry at 20 (open bars), 40 (hatched bars), and 60 (closed bars) min. For CHS versus corresponding EGTA treatment time points, P < 0.05 (*) or P < 0.01 (**); n = 3 or 4. Error bars indicate standard deviations.

FIG. 6.

Role of complement and Fc receptors in neutrophil-adenovirus interaction. (A) Cy2-AdLuc was incubated with neutrophils in the presence of blocking CD35, CD11b, CD18, or isotype control antibodies. Cells were analyzed by flow cytometry at 20 (open bars), 40 (hatched bars), and 60 (closed bars) min. For anti-CD35 versus isotype control, P < 0.05 (*); n = 3 to 5. (B) Cy2-AdLuc was incubated with neutrophils in the presence of blocking CD32, CD16, mouse IgG1, or mouse IgG2a antibodies. Cells were analyzed by flow cytometry at 40 min. For treatment groups versus IgG₁ control, P < 0.05 (*) and P < 0.01 (**); n = 6. For combined Fc receptor blockade versus individual blockade, P = not significant (NS). (C) Effect of combined complement and Fc receptor blockade. Cy2-AdLuc was incubated with neutrophils in the presence of blocking CD35, CD32, CD16, mouse IgG2a, or control mouse IgG1 antibodies. Cells were analyzed by flow cytometry at 40 min. For CD35 blockade or combined receptor blockade versus IgG1 control, P < 0.05 (*) and P < 0.005 (***), respectively; n = 6. For combined CD35 and Fc receptor blockade versus CD35 alone, P < 0.05 (*); n = 6. Error bars indicate standard deviations.