Insoluble and soluble immune complexes activate neutrophils by distinct activation mechanisms: changes in functional responses induced by priming with cytokines

Abstract

Background: Rheumatoid synovial fluid contains both soluble and insoluble immune complexes that can activate infiltrating immune cells such as neutrophils.

Objectives: To determine if these different complexes activate neutrophils through similar or different receptor signalling pathways. In particular, to determine the circumstances which result in the secretion of tissue damaging reactive oxygen metabolites and granule enzymes.

Methods: Blood neutrophils were incubated with synthetic soluble and insoluble immune complexes and the ability to generate reactive oxidants tested by luminescence or spectrophotometric assays that distinguished between intracellular and extracellular production. Degranulation of myeloperoxidase and lactoferrin was determined by western blotting. The roles of FcγRII (CD32) and FcγRIIIb (CD16) were determined by incubation with Fab/F(ab`)₂ fragments before activation. The effect of cytokine priming was determined by incubation with GM-CSF.

Results: Insoluble immune complexes activated unprimed neutrophils, but most of the oxidants produced were intracellular. This activation required FcγRIIIb, but not FcγRII function. Soluble complexes failed to activate unprimed neutrophils but generated a rapid and extensive secretion of reactive oxygen metabolites when the cells were primed with granulocyte-macrophage colony stimulating factor (GM-CSF). This activity required both FcγRII and FcγRIIIb function. Insoluble immune complexes activated the release of granule enzymes from primed or unprimed neutrophils, but the kinetics of release did not parallel those of secretion of reactive oxygen metabolites. Only primed neutrophils released enzymes in response to soluble complexes.

Conclusions: Soluble and insoluble immune complexes activate neutrophils by separate receptor signalling pathways. Profound changes in neutrophil responsiveness to these complexes occur after cytokine priming.

Full Text

The Full Text of this article is available as a PDF (207.6 KB).

Figure 1 .

Activation of the neutrophil respiratory burst by immune complexes. (A and B) Neutrophils were incubated in HBSS at 5x10⁵ cells/ml in medium that was supplemented with either 10 µM luminol or 10 µM isoluminol. (C) Cells were incubated at 10⁶ cells/ml in HBSS supplemented with 75 µM cytochrome c. Neutrophils were incubated for 45 minutes in the absence (unprimed) or presence (primed) of GM-CSF (50 U/ml). They were then stimulated with either soluble or insoluble immune complexes, as described in "Materials and methods". Typical result of at least six separate experiments are shown.

Figure 2 .

Effect of oxidant scavengers on chemiluminescence: activation by soluble immune complexes. GM-CSF primed neutrophils were incubated at 5x10⁵ cells/ml in HBSS supplemented with either 10 µM luminol (A) or 10 µM isoluminol (B), in the absence or presence of SOD, catalase, or sodium azide, as described in "Materials and methods". At time zero, cells were stimulated by the addition of soluble immune complexes. Typical results of at least six separate experiments are shown.

Figure 3 .

Effect of oxidant scavengers on chemiluminescence: activation by insoluble immune complexes. GM-CSF primed neutrophils were incubated at 5x10⁵ cells/ml in HBSS supplemented with either 10 µM luminol (A) or 10 µM isoluminol (B), in the absence or presence of SOD, catalase, or sodium azide, as described in "Materials and methods". At time zero, cells were stimulated by the addition of insoluble immune complexes. Typical results of at least six separate experiments are shown.

Figure 4 .

Effect of FcγR blocking on neutrophil activation by immune complexes. GM-CSF primed neutrophils were incubated in the absence or presence of either an F(ab`)₂ fragment of 3G8 (anti-FcγRIIIb) or an Fab fragment of IV.3 (anti-FcγRII) or with both fragments, as described in "Materials and methods". Luminol chemiluminescence was then measured after the addition of either soluble (A) or insoluble (B) immune complexes. Typical results of at least six separate experiments are shown.

Figure 5 .

Effect of FcγRIIIb depletion on neutrophil activation by immune complexes. GM-CSF primed neutrophils were incubated in the absence or presence of PI-PLC as described in "Materials and methods". They were then stimulated by the addition of either soluble (A) or insoluble (B) immune complexes and luminol chemiluminescence measured. Typical results of at least six separate experiments are shown.

Figure 6 .

Activation of degranulation by immune complexes. Neutrophils were incubated in the presence and absence of GM-CSF and then stimulated for 45 minutes by the addition of either soluble or insoluble immune complexes. Cell-free supernatants were then analysed for the presence of either lactoferrin (A) or myeloperoxidase (B) by immunoblotting. In (A) samples were loaded as follows: 1, unprimed neutrophils, no additions; 2, unprimed neutrophils, soluble immune complexes; 3, unprimed neutrophils, insoluble immune complexes; 4, primed neutrophils, soluble immune complexes; 5, primed neutrophils insoluble immune complexes; 6, primed neutrophils no additions. In (B) samples were loaded as follows: 1–4, unprimed neutrophils; 5–8, primed neutrophils; 1, 5, control, no additions; 2, 6, phorbol myristate acetate (0.1 µg/ml); 3, 7, insoluble immune complexes; 4, 8, soluble immune complexes. Typical results of at least six separate experiments are shown.

Figure 7 .

Time course of degranulation in response to immune complexes. Neutrophils were primed by incubation in the presence and absence of GM-CSF and then incubated in the absence (control, not shown) or presence of either insoluble or soluble immune complexes. At time intervals of up to 45 minutes, cell-free supernatants were tested for the presence of either myeloperoxidase (A) or lactoferrin (B) by western blotting. Typical results of at least three separate experiments are shown.