Martti Laan

Endogenous IL-17 as a mediator of neutrophil recruitment caused by endotoxin exposure in mouse airways

We have previously demonstrated that administration of the recently described cytokine IL-17 in rat airways in vivo recruits and activates neutrophils locally. In the current study, we examined whether endogenous IL-17 is involved in mediating neutrophil recruitment caused by endotoxin exposure in mouse airways. Our in vivo data show that local endotoxin exposure causes the release of free, soluble IL-17 protein 6 h later. Systemic pretreatment with a neutralizing anti-IL-17 Ab almost completely inhibits neutrophil recruitment 24 h, but not 6 h, after endotoxin exposure in the airways. Pretreatment with neutralizing anti-IL-6 and anti-macrophage inflammatory protein (MIP)-2 Abs inhibits neutrophil recruitment caused by local endotoxin exposure and IL-17, respectively. Our in vitro data show that endotoxin exposure stimulates the release of soluble IL-17 protein in T lymphocytes harvested from lung and spleen, respectively, and that this cytokine release requires coculture with airway macrophages. Intracellular IL-17 protein is detected in T lymphocytes from spleen but not in airway macrophages after coculture and stimulation of these two cell types. Finally, anti-IL-17 does not alter endotoxin-induced release of IL-6 and MIP-2 from T lymphocytes and airway macrophages in coculture. In conclusion, our results indicate that endotoxin exposure causes the release of IL-17 from T lymphocytes and that this cytokine release requires the presence of macrophages. Once released, endogenous IL-17 acts in part by inducing local release of neutrophil-mobilizing cytokines such as IL-6 and MIP-2, from nonlymphocyte, nonmacrophage cells, and this contributes to recruitment of neutrophils in the airways. These IL-17-related mechanisms constitute potential targets for pharmacotherapy against exaggerated neutrophil recruitment in airway disease.

IL-17-induced cytokine release in human bronchial epithelial cells in vitro : role of mitogen-activated protein (MAP) kinases

Recent data indicate that interleukin (IL)‐17 may contribute to neutrophilic airway inflammation by inducing the release of neutrophil‐mobilizing cytokines from airway cells. The aim of this study was to evaluate the role of mitogen activated protein kinases in IL‐17 induced release of IL‐8 and IL‐6 in bronchial epithelial cells. Transformed human bronchial epithelial cells (16HBE) were stimulated with either IL‐17 or vehicle. Both groups were treated either with SB202190 (inhibitor of p38 MAP kinase), PD98059 (inhibitor of extracellular‐signal‐regulated kinase [ERK] pathway), Ro‐31‐7549 (protein kinase C [PKC] inhibitor), LY 294002 (a phosphatidylinositol 3‐kinase [PI 3‐kinase] inhibitor) or vehicle. IL‐6 and IL‐8 levels were measured in conditioned media by ELISA. The IL‐17‐induced release of IL‐6 and IL‐8 was concentration‐dependently inhibited by SB202190 and by PD98059 in bronchial epithelial cells without affecting cell proliferation or survival. Ro‐31‐7549 and LY294002 had no significant effect on IL‐17‐induced IL‐6 or IL‐8 release in bronchial epithelial cells. Taken together, these data indicate a role for p38 and ERK kinase pathways in IL‐17‐induced release of neutrophil‐mobilizing cytokines in human bronchial epithelial cells. These mechanisms constitute potential pharmacotherapeutical targets for inhibition of the IL‐17‐mediated airway neutrophilia.

Pharmacological modulation of interleukin-17-induced GCP-2-, GRO-α- and interleukin-8 release in human bronchial epithelial cells

The cytokine interleukin-17 may play a role in the recruitment of airway neutrophils, and interleukin-17 protein is increased in the airways of patients with asthma. In this study, we characterised the effect of interleukin-17 on the release of the neutrophil-recruiting cytokines granulocyte chemotactic protein (GCP)-2, growth-related oncogene (GRO)-alpha and interleukin-8 in human bronchial epithelial (HBE) cells. We also characterised the involvement of mitogen-activated protein (MAP) kinases as well as the effect of beta-adrenoceptor and glucocorticoid receptor stimulation and calcineurin and P-glycoprotein inhibition on these epithelial responses to interleukin-17. We found that interleukin-17 (1-1000 ng/ml) increased the release of GCP-2, GRO-alpha and interleukin-8 in a concentration-dependent manner. This interleukin-17-induced release of C-X-C chemokines was sensitive to inhibition of the p38 MAP kinase pathway and to stimulation of glucocorticoid receptors. In contrast, stimulation of beta-adrenoceptors increased the release of interleukin-8 and did not markedly alter the release of GCP-2 and GRO-alpha. Inhibition of calcineurin and of P-glycoproteins did not exert any substantial effect on the release of C-X-C chemokines. In conclusion, interleukin-17 bears the potential to increase neutrophil recruitment into the airways by releasing several, different C-X-C chemokines, including GCP-2, GRO-alpha and interleukin-8 in human bronchial epithelial cells. Inhibition of the p38 MAP kinase pathway and glucocorticoid receptor stimulation constitute two credible therapeutic strategies against this interleukin-17-induced release of neutrophil-recruiting cytokines.

A role of GM-CSF in the accumulation of neutrophils in the airways caused by IL-17 and TNF‐α

The T‐cell cytokine interleukin (IL)-17 selectively accumulates neutrophils in murine airways in vivo and may thus constitute a link between activation of T‐lymphocytes and accumulation of neutrophils. In this study, the authors evaluated the role of granulocyte macrophage-colony stimulating factor (GM-CSF) in accumulation of neutrophils in the airways caused by IL-17 and tumour necrosis factor (TNF)‐α. In vitro, human (h) IL-17 concentration-dependently stimulated the release of GM-CSF protein (enzyme-linked immunosorbent assay) in human bronchial epithelial cells (16HBE). IL-17 also time-dependently stimulated the release of GM-CSF protein in venous endothelial (human umbilical vein endothelial cells) cells in vitro. Co-stimulation with IL-17 plus the pro-inflammatory cytokine TNF‐α potentiated the release of GM-CSF protein in 16HBE cells. hIL-17 also enhanced the expression of GM-CSF messenger ribonucleic acid in 16HBE cells (reverse transcriptase polymerase chain reaction), with a similar order of magnitude as TNF‐α. Conditioned cell medium from bronchial epithelial cells co-stimulated with hIL-17 plus TNF‐α prolonged survival (trypan blue exclusion) of human neutrophils in vitro and this effect was blocked by an anti-GM-CSF antibody. In vivo, local co-stimulation with mouse IL-17 plus TNF‐α caused an additive potentiation of the accumulation of neutrophils in bronchoalveolar lavage fluid from mouse airways and this effect was blocked by an anti‐GM-CSF antibody given systemically. In conclusion, granulocyte macrophage-colony stimulating factor is involved in the accumulation of neutrophils in the airways caused by interleukin-17 and tumour necrosis factor‐α, probably via effects on both recruitment and survival of neutrophils.

Free, soluble interleukin-17 protein during severe inflammation in human airways

Studies in rodents indicate that the cytokine, interleukin (IL)-17, links the activation of T-lymphocytes to neutrophilic inflammation. The aim of the current study was to determine whether free, soluble IL-17 protein can be released during severe inflammation in human airways. Fifteen healthy subjects were exposed to a swine confinement in order to induce severe inflammation characterized by high neutrophil numbers in the airways. Bronchoalveolar lavage (BAL) fluid was harvested 2 weeks prior to and 24u2005h after this exposure and the concentration of IL-17 protein was measured using an enzyme-linked immunosorbent assay. Total and cell differential counts were also performed in BAL fluid. Prior to exposure to the swine confinement, the concentration of IL-17 in BAL fluid was low (<7.8u2005pg·mL−1) in 14 out of 15 subjects. However, exposure to the swine confinement caused an increase in IL-17 in 13 out of 15 subjects (median IL-17 concentration of 26.9u2005pg·mL−1). This exposure also caused a 51-fold increase in the concentration of neutrophils in BAL fluid. To conclude, free, soluble interleukin-17 protein can be released during severe inflammation characterized by high neutrophil numbers in human airways. The significance of interleukin-17 in inflammatory airway diseases therefore deserves further evaluation.

IL-17 as a potential target for modulating airway neutrophilia.

Several chronic inflammatory airway diseases are characterized by an increased number of neutrophils in the airways. There is evidence that the recruitment of these neutrophils can be controlled by certain T-lymphocytes. However, the mechanisms behind this T-cell control of airway neutrophilia are poorly understood. In this review, we summarize the evidence that interleukin (IL)-17 released from T-lymphocytes of the CD45RO+ subset can link the activation of these T-cells to the recruitment and activation of neutrophils. This evidence suggests that pharmacotherapeutical modulation of neutrophilic airway inflammation can be achieved using several different strategies, including inhibition of IL-17 production by cAMP elevating agents or certain nuclear factor inhibitors, neutralization of released IL-17 protein by specific anti-IL-17-antibodies, blockade of the IL-17 receptor as well as inhibition of certain MAP kinases mediating the post receptor effects of IL-17 in airway cells. Additional studies on animals in vivo and patients, respectively, are needed to further evaluate the pharmacotherapeutical potential of these strategies.

IL-16 in the airways of lung allograft recipients with acute rejection or obliterative bronchiolitis

Acute rejection (AR) is the principal risk factor for obliterative bronchiolitis (OB), the major complication of lung transplantation. It is known that activated CD4+u2003T lymphocytes are involved in the development of AR and that interleukin (IL)‐16 can inhibit the activity of CD4+u2003T lymphocytes. In this study, we evaluated whether the concentration of IL‐16 in the airways is altered in AR or OB and, if so, how this IL‐16 concentration relates to the number or activity of airway lymphocytes. The concentration of IL‐16 protein was measured in bronchoalveolar lavage (BAL) fluid at three time‐points in lung allograft recipients with either AR or OB and in matched controls using ELISA. The concentration of soluble IL‐2 receptor (R) protein was measured in BAL fluid using ELISA as well, as an indicator of lymphocyte activity. The percentage of airway lymphocytes was evaluated by performing BAL differential cell counts. Lung allograft recipients with AR displayed lower IL‐16 concentrations compared with matched control patients and this IL‐16 concentration correlated negatively with the sIL‐2R concentration, but it did not correlate with the percentage of lymphocytes in BAL fluid. In contrast, in BAL fluid from lung allograft recipients with OB, the IL‐16 concentration was not altered compared with matched control patients and it did not correlate with the percentage of lymphocytes or with the sIL‐2R concentration. These data are compatible with an increase in IL‐16 playing a protective role against AR but not against OB and, hypothetically, this type of protective effect could be exerted via a down‐regulation of the activity of T lymphocytes.

Impact of tobacco smoke on interleukin-16 protein in human airways, lymphoid tissue and T lymphocytes

CD4+u2003and CD8+ lymphocytes are mobilized in severe chronic obstructive pulmonary disease (COPD) and the CD8+ cytokine interleukin (IL)‐16 is believed to be important in regulating the recruitment and activity of CD4+ lymphocytes. In the current study, we examined whether tobacco smoke exerts an impact not only on IL‐16 in the lower airways but also in CD4+ or CD8+ lymphocytes or in lymphoid tissue. The concentration of IL‐16 protein was measured by enzyme‐linked immunosorbent assay (ELISA) in concentrated bronchoalveolar lavage fluid (BALF) collected from 33 smokers with chronic bronchitis (CB), eight asymptomatic smokers (AS) and seven healthy never‐smokers (NS). The concentrations of IL‐16 and soluble IL‐2 receptor alpha (sIL‐2Rα) protein were also measured in conditioned medium from human blood CD4+ and CD8+ lymphocytes stimulated with tobacco smoke extract (TSE) in vitro. IL‐16 mRNA was assessed in vitro as well, using reverse transcription–polymerase chain reaction (RT‐PCR). Finally, the intracellular immunoreactivity for IL‐16 protein (IL‐16IR) was assessed in six matched pairs of palatine tonsils from smokers and non‐smokers. BALF IL‐16 was higher in CB and AS than in NS. TSE substantially increased the concentration of IL‐16 but not sIL‐2Rα in conditioned medium from CD4+ and CD8+ lymphocytes. There was no corresponding effect on IL‐16 mRNA. IL‐16IR in tonsils was lower in smokers than in non‐smokers. The current findings demonstrate that tobacco smoke exerts a wide impact on the CD8+ cytokine IL‐16, in the airway lumen, in blood CD4+ and CD8+ lymphocytes and in lymphoid tissue. The effect on IL‐16 release may be selective for preformed IL‐16 in CD4+ lymphocytes. New clinical studies are required to evaluate whether tobacco smoke mobilizes T lymphocytes via IL‐16 in the lower airways and whether this mechanism can be targeted in COPD.

The IL-17 Family of Cytokines - Applications in Respiratory Medicine and Allergology

The excessive accumulation of granulocytes is believed to constitute an important factor in inflammatory airway diseases, including asthma and chronic obstructive pulmonary disease (COPD). Notably, T helper (Th) cells are known to produce cytokines that are involved in the mobilization of eosinophils and neutrophils. Currently, it is believed that a third population of Th cells, the recently described Th17 population, accounts for the production of several members of the interleukin (IL)-17 family of cytokines. The members of this cytokine family have proven abilities to recruit and activate neutrophils and eosiniphils. This review summarizes the evidence that these cytokines constitute key mediators of the Th-controlled granulocyte influx in airway disease and points out molecular target candidates for therapy as well as related patents.