Evidence for chemokine synergy during neutrophil migration in ARDS

Abstract

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterised by pulmonary oedema, respiratory failure and severe inflammation. ARDS is further characterised by the recruitment of neutrophils into the lung interstitium and alveolar space.

Objectives: The factors that regulate neutrophil infiltration into the inflamed lung and our understanding of the pathomechanisms in ARDS remain incomplete. This study aimed at determining the role of the chemokine (C-C motif) ligand (CCL)2 and CCL7 in ARDS.

Methods: CCL2 and CCL7 protein levels were measured in bronchoalveolar lavage (BAL) fluid obtained from lipopolysaccharide(LPS)-challenged human volunteers and two separate cohorts of patients with ARDS. Neutrophil chemotaxis to ARDS BAL fluid was evaluated and the contribution of each was assessed and compared with chemokine (C-X-C motif) ligand 8 (CXCL8). Chemokine receptor expression on neutrophils from blood or BAL fluid of patients with ARDS was analysed by flow cytometry.

Results: CCL2 and CCL7 were significantly elevated in BAL fluid recovered from LPS-challenged volunteers and patients with ARDS. BAL fluid from patients with ARDS was highly chemotactic for human neutrophils and neutralising either CCL2 or CCL7 attenuated the neutrophil chemotactic response. Moreover, CCL2 and CCL7 synergised with CXCL8 to promote neutrophil migration. Furthermore, neutrophils isolated from the blood or BAL fluid differentially regulated the cell surface expression of chemokine (C-X-C motif) receptor 1 and C-C chemokine receptor type 2 during ARDS.

Conclusion: This study highlights important inflammatory chemokines involved in regulating neutrophil migration, which may have potential value as therapeutic targets for the treatment of ARDS.

Keywords: ARDS; Cytokine Biology; Innate Immunity; Neutrophil Biology.

Figure 1

Plots showing th elevated bronchoalveolar lavage (BAL) fluid levels of chemokine (C-C motif) ligand (CCL)2 and CCL7 in a human model of acute lung inflammation and in patients with acute respiratory distress syndrome (ARDS). Healthy volunteers were challenged with 0.6% nebulised saline (n=5) or with 50 μg of nebulised lipopolysaccharide(LPS) (n=25) and BAL was performed 6 hours later. The levels of CCL2 (A) and CCL7 (B) were measured in BAL fluid recovered from saline and LPS-challenged volunteers by ELISA (right axis). BAL was also performed on patients with a clinical diagnosis of ARDS and the levels of CCL2 (A) and CCL7 (B) were measured (n=18) in recovered ARDS BAL fluid by ELISA (left axis). BAL fluid was also recovered from a second cohort of patients who underwent surgical oesophagectomy who were either at risk of developing ARDS (n=20) or who went on to develop ARDS (n=20). The levels of CCL2 (C) and CCL7 (D) were measured in recovered BAL fluid by ELISA. Power calculations were based on previous studies with α=0.05; power=0.8; difference between means=1.5 (50% reduction); SD=1.6. Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.

Figure 2

Chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand (CCL)2 and CCL7 contribute to the neutrophil chemotactic activity of acute respiratory distress syndrome (ARDS) bronchoalveolar lavage fluid (BALF). Human neutrophils were isolated from the blood of healthy volunteers and chemotaxis was measured across 3 µm membranes (ChemoTX, NeuroProbe) in response to media alone, healthy volunteer BALF (n=11) or BALF from patients with ARDS (n=18) (A). Neutrophil chemotaxis was measured as the chemotactic index (number of migrated neutrophils following treatment vs medium control) after 1 hour (healthy BALF, left axis; ARDS BALF, right axis). Neutrophil chemotaxis towards BALF obtained from patients with ARDS was measured in the presence of polyclonal Ig control (20 μg/mL), neutralising anti- CXCL8) (10 μg/mL), anti-CCL2 (10 μg/mL) and a combination (10 μg/mL of each) of the two antibodies (B) or anti-CXCL8 (10 μg/mL), anti-CCL7 (10 μg/mL) and a combination (10 μg/mL of each) of the two antibodies (C). Antibodies were incubated in the presence of ARDS BALF for 20 min prior to the addition of isolated neutrophils. Differences in neutrophil chemotaxis between treatment groups were measured as the percentage of maximal chemotaxis to ARDS BALF alone (n=18 ARDS BALF samples using neutrophils isolated from multiple healthy human volunteers). Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.

Figure 3

Human neutrophils migrate toward recombinant chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-C motif) ligand (CCL)2 and CCL7. Human neutrophils were isolated from the blood of healthy volunteers and chemotaxis was measured across 3 µm membranes (ChemoTX, NeuroProbe) in response to recombinant human CXCL8, CCL2 and CCL7 (A), over a range of concentrations as stated. Neutrophil chemotaxis was also measured in response to a suboptimal concentration of recombinant human CXCL8 (5 ng/mL) in combination with recombinant human CCL2 (B) or recombinant human CCL7 (C) across a range of concentrations as stated (n=3 independent experiments using different healthy volunteer donors). Neutrophil chemotaxis was measured as the chemotactic index (number of migrated neutrophils following treatment vs medium control) after 1 hour. Statistical analysis was performed using one-way analysis of variance with Newman-Keuls post-hoc test.

Figure 4

Neutrophils differentially regulate chemokine receptors during acute respiratory distress syndrome (ARDS). Bronchoalveolar lavage fluid (BALF) and blood was recovered from patients with a clinical diagnosis of ARDS (patient details in online supplementary table S2). Flow cytometry analysis of neutrophils was performed by gating on CD16+, CD14−, human leucocyte antigen-D-related (HLA-DR)− leucocyte populations. The neutrophil population was further verified by FSC and SSC properties (details in online supplementary figure S2). The expression of chemokine (C-X-C motif) receptor (CXCR)1 (A), CXCR2 (B), C-C chemokine receptor (CCR)1 (C), CCR2 (D) and CCR3 (E) on neutrophils from the blood of patients with ARDS was compared with neutrophils from the BALF of matching patients with ARDS (n=10). Data are represented as the ratio of mean fluorescent intensity (MFI) of each chemokine receptor expressed on BALF neutrophils versus blood neutrophils (direct measurements of MFI are presented in online supplementary figure S3). Statistical analysis was performed using a Wilcoxon signed rank test. FSC, forward scatter; SSC, side scatter.