The role of interleukin-1beta in direct and toll-like receptor 4-mediated neutrophil activation and survival

Abstract

The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1beta has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1beta fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1beta induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1beta, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1(-/-) mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1beta on neutrophils are indirect and mediated by other cells.

Figure 1

IL-1β does not modulate neutrophil apoptosis in the absence of PBMCs. A: Highly purified neutrophils were stimulated with IL-1β (0.1 to 10 ng/ml; filled squares), IL-18 (0.1 to 100 ng/ml; open circles), or pLPS (1 ng/ml; asterisks) for 4 and 22 hours, and rates of apoptosis were determined by morphology. B: In a separate series of experiments cells were stimulated with these agonists and rates of apoptosis were determined by binding of Annexin V, measured by flow cytometry. Charts show mean ± SEM data from four independent experiments. C: PBMCs (5%) were added back to populations of highly purified neutrophils and treated with IL-1β for 22 hours. Apoptosis was quantified by morphology (n = 7, ±SEM, analyzed versus buffer-treated control using a paired t-test in which an asterisk indicates P < 0.05). D: Highly pure neutrophils (HRN0) were treated with IL-1β in the presence or absence of 5% PBMCs, and apoptosis quantified by Annexin V binding (n = 4, ±SEM; *, P < 0.05 for effect of PBMCs on responses to 100 ng/ml of IL-1β, two-way analysis of variance with Bonferroni’s post test).

Figure 2

IL-1β is unable to modulate neutrophil adhesion molecule expression. Highly purified neutrophils were treated with the indicated concentrations of IL-1β, pLPS, or fMLP. Expression of L-selectin and CD11b was determined by flow cytometry and data was expressed relative to the geometric mean fluorescence intensity of buffer-treated controls. Top: Representative histograms of L-selectin (A) and CD11b (B) expression after activation by the indicated agonists. The control histogram is binding of an isotype-matched control antibody; buffer indicates cells treated with buffer at 37°C before measurement of CD11b and L-selectin expression. C: Mean data (n = 3, ±SEM).

Figure 3

IL-1β is unable to enhance neutrophil IL-8 generation. Highly purified neutrophils were stimulated for 24 hours with a concentration range of IL-1β (0.1 to 100 ng/ml), pLPS (0.1 to 1 ng/ml), or medium alone, either in the presence (filled bars) or absence (open bars) of 5% PBMCs. Cell-free supernatants were prepared and IL-8 measured by enzyme-linked immunosorbent assay. IL-1β was unable to significantly enhance IL-8 generation in any condition tested. Data shown are mean ± SEM from three independent experiments. *, P < 0.05 indicates statistically significant change in IL-8 production within indicated pair, analyzed by two-way analysis of variance and Bonferroni’s post test.

Figure 4

pLPS pretreatment does not render neutrophils sensitive to anti-apoptotic effects of IL-1β. Highly purified neutrophils were pretreated with medium alone or pLPS (1 ng/ml) for 4 hours before the addition of a selection of cytokines comprising of IL-1β (10 ng/ml), GM-CSF (50 U/ml), and IL-18 (100 ng/ml) for a further 18 hours. Apoptosis was assessed by light microscopy. Data shown are means (±SEM) from four independent experiments (***, P < 0.001 indicates change compared to medium alone; ^§§§, P < 0.001 indicates change in populations pretreated with pLPS, analyzed by analysis of variance with Tukey’s post test).

Figure 5

Soluble IL-1ra does not prevent pLPS-mediated survival. Highly purified neutrophils were pretreated with medium alone or IL-1ra (1 mg/ml shown here, lower concentrations were also found to be ineffective) for 4 hours before the addition of pLPS (1 ng/ml) for a further 22 hours. Apoptosis was assessed by light microscopy. Data shown are means (±SEM) from three independent experiments (**, P < 0.01 indicates change compared to medium alone, analyzed by two-way analysis of variance with Bonferroni’s post test).

Figure 6

IL-1R1^−/− neutrophils exhibit delayed apoptosis with LPS. Neutrophils isolated from IL-1R1^−/− mice were cultured with cLPS (1 μg/ml) for 6 (A) and 16 (B) hours. RAW 264.7 cells (10%) were added to 16-hour cultures. Apoptosis was assessed by light microscopy. Data shown are means from three independent experiments. A: **, Significant difference of P < 0.01 between medium and LPS-treated cells, determined by t-test. B: *, Significant difference of P < 0.05 between indicated conditions, analyzed by analysis of variance and Tukey’s post test.

Figure 7

Compartmentalization of neutrophil regulation. Neutrophil recruitment is under the control of tissue cells, whose cytokines cause up-regulation of endothelial adhesion molecule expression and provide directional migration cues (A). At sites of inflammation, mediators such as LPS activate the cell (B), but do not prolong life span to the same degree. Thus, the ongoing need for a surviving population of neutrophils may again be determined by tissue cells or monocytes (C), whose activation is in turn regulated. Ultimately, resolution of neutrophilic inflammation proceeds by apoptosis (D). IL-1 may play roles in aiding neutrophil recruitment by actions on endothelium, and as a mediator activating cells such as monocytes to produce neutrophil survival factors, perhaps in autocrine loops, but does not appear to have a major direct role in regulating neutrophil survival.