Abstract
It is now established that an excessive and sustained mobilisation of neutrophils is a hallmark of several chronic inflammatory lung disorders, including severe obstructive lung disease. This article reviews evidence that the cytokine interleukin (IL)-17A is a major orchestrator of sustained neutrophilic mobilisation.
Current evidence suggests that IL-17A is produced by T-lymphocytes, and that it exerts an orchestrating effect on the accumulation and associated activity of neutrophils in the bronchoalveolar space indirectly, through an induced release of specific cytokines and colony-stimulating factors in resident lung cells.
Although the involvement of IL-17A in inflammatory lung disorders is supported by several recent studies, its causative role is still uncertain.
However, the unique position of interleukin-17A at the interface between acquired and innate immunity puts this cytokine forward as an important signal for the reinforcement of host defence; it also implies that interleukin-17A may constitute a useful target for pharmacotherapeutic intervention.
The T-lymphocyte cytokine interleukin (IL)-17(A) was discovered in 1993, and, since the first published report on the neutrophil-accumulating effect of IL-17A in the bronchoalveolar space of rats in 1999 1–3, research in this area has generated a growing body of evidence suggesting that IL-17A plays a central role in host defence within the lungs. IL-17A exerts this effect by orchestrating the local release of neutrophil-mobilising factors in resident cells (table 1⇓). In the present review article, the current evidence that neutrophils are important players in chronic inflammatory lung disorders and that IL-17A is involved in coordinating neutrophil accumulation in these disorders is presented. The review also addresses the potential therapeutic utility of drugs targeting IL-17A and its receptor(s).
Functional characteristics of interleukin(IL)-17A in lungs
NEUTROPHILS AND INNATE IMMUNITY
Neutrophils are believed to have evolved as first-line defenders against a wide range of pathogens, and this is particularly true for the lungs 18–23. Functionally intact neutrophils are essential for life in mammals, including humans. Patients suffering from defects in neutrophil function, such as their formation in bone marrow, adhesion to inflamed blood vessels, extravasation, accumulation in tissue, or neutrophil oxidant and proteolytic effector functions, suffer from recurrent and severe infections 18–23. It is noteworthy that neutrophil mobilisation is one of the earliest and most consistent consequences in the immune system after activation of pattern recognition receptors, in particular Toll-like receptors, by bacteria and other invading organisms 23–27. Given the crucial importance of neutrophils to host defence, it is not surprising that these pathways mediate the expression of a wide range of different neutrophil-mobilising factors, including chemotactic cytokines (IL-8 and other CXC chemokines), activating factors (IL-6) and colony-stimulating factors (granulocyte (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF)) 28, 29.
ACCUMULATION OF NEUTROPHILS IN LUNG DISEASE
Beside its utility in host defence, the potent molecular armature that equips the neutrophil to destroy pathogens is also potentially harmful, if turned against host tissue. In line with such a destructive action, the accumulation of neutrophils locally in upper and lower airways appears to be linked to the long-term course and exacerbations of several acute and chronic lung disorders, including acute respiratory distress syndrome (ARDS), asthma, bronchiectasis, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and chronic lung allograft rejection 30–58. The accumulation of neutrophils in the airways can also be linked to functional anomalies such as nonspecific bronchial hyperreactivity, hypersecretion and cough, all of which are key characteristics of obstructive lung disease 59, 60. This evidence provides a solid rationale for focusing research on the role of mechanisms leading to accumulation of neutrophils in severe asthma and COPD. As there are currently no specific means of safely suppressing neutrophils in the lungs of humans, the case for the neutrophil as a pathogenic factor is based largely upon association studies. The evidence includes increased numbers of neutrophils being mainly present in airway tissue, bronchoalveolar lavage fluid or induced sputum.
ACCUMULATION OF NEUTROPHILS AND LUNG FUNCTION
A functional impact of neutrophils is supported by the observation that the number of neutrophils in the bronchoalveolar space correlates with the degree of nonspecific bronchial hyperreactivity in patients with mild, as well as severe persistent, asthma 34, 60. In severe persistent asthma, the bronchoalveolar content of the neutrophilic enzyme myeloperoxidase (MPO) also correlates with the degree of nonspecific bronchial hyperreactivity 34. Interestingly, the induced release of MPO from isolated neutrophils originating from the blood of patients with asthma correlates negatively with lung function (forced expiratory volume in one second), suggesting a systemic impact, and that neutrophil products may constitute surrogate biomarkers of more severe disease 61. By analogy, the number of neutrophils in the bronchoalveolar space and the mucosa, respectively, also correlate negatively with lung function in smokers, with or without chronic bronchitis and COPD 39, 44–46. In addition, among tobacco smokers, the number of luminal neutrophils correlates positively with the annual decline in lung function 59. It is also known that neutrophil recruitment is associated with bronchial hyperresponsiveness in guinea pig airways in vivo 62. Interestingly, a recent study on patients with mild stable asthma claimed that treatment with a β-adrenoceptor agonist (but not a glucocorticoid) results in a decrease in the number of neutrophils within the bronchoalveolar space in parallel with a decrease in nonspecific bronchial reactivity and an improvement in subjective symptoms 63. Taken together, the clinical evidence from patients with obstructive lung disorders suggests that neutrophils constitute an important pathogenic factor, in particular for the long-term course and exacerbations in severe asthma and COPD 30–33, 39–47.
NEUTROPHIL PRODUCTION OF COMPOUNDS WITH PATHOGENIC POTENTIAL
Hypothetically, neutrophils might cause tissue damage through the release of several protein-degrading or cytotoxic compounds.
Neutrophil elastase
There is substantial evidence suggesting that neutrophil elastase degrades elastin, a key structural lung component that prevents small airways from collapsing, and that this leads to an emphysema-like condition in the lungs, and possibly also to remodelling of airway tissue 64–68. Human neutrophil elastase also augments the fibroblast-mediated contraction of collagen gels in vitro, an observation underlining the potential remodelling capacity of this serine proteinase 68, 69. Interestingly, neutrophil elastase appears to substantially contribute to secretion in airway gland cells from humans and other mammals in vitro, as well as in the upper airways of dogs in vivo 70–72. Neutrophil elastase can also cause nonspecific bronchial hyperresponsiveness in guinea pig airways in vivo 64, 65. There is even data from in vitro studies demonstrating that peptide fragments from human elastin that has been degraded using human neutrophil elastase cause chemotaxis of human blood monocytes and calf skin fibroblasts; evidence compatible with this elastase causing the recruitment of cells other than neutrophils and thus perpetuating inflammation at sites of neutrophil accumulation 73, 74. Furthermore, neutrophils also contain another elastolytic enzyme, proteinase-3, which may cause effects similar to those of neutrophil elastase 75–77.
In view of the experimental evidence regarding pathogenically relevant effects exerted by neutrophil elastase, the local detection of this compound in human lung disease is of substantial interest. Thus, in the airway submucosa of patients with severe asthma, neutrophil elastase is detected regardless of whether eosinophils are present or not, thus showing neutrophil activity to be a more consistent factor than eosinophils, in terms of association with severe asthma 35. The concentration of neutrophil elastase is also increased in the bronchoalveolar space during severe asthma, even when no bacterial infection is detected 31–33. In the same compartment, an increased concentration of neutrophil elastase is also present in ARDS, chronic bronchitis, COPD and CF 32, 51, 78–80. It is noteworthy that several clinical studies demonstrate a correlation between the local concentration of elastase and lung function; these studies include patients with asthma, chronic bronchitis and CF 32, 81.
Matrix metalloproteinases
It is well known that neutrophils can release proteolytic enzymes with tissue-degrading capacity other than neutrophil elastase and proteinase-3. Thus, the two matrix metalloproteinases (MMPs) MMP-8 and -9 are released from human neutrophils upon induced activation in vitro, regardless of whether the donors are healthy subjects or patients, with asthma or COPD 82–84. In the bronchoalveolar space of patients with severe asthma, in particular, the intracellular level of active MMP-8 is increased and seems mainly to be localised in neutrophils 76. In addition, the concentration of MMP-9 is increased in the bronchoalveolar space of patients with asthma, particularly in patients with severe disease, and the activity of this form of MMP-9 may be less sensitive to a glucocorticoid among patients with asthma than among healthy control subjects 56, 85. In the same compartment, the concentrations of free soluble MMP-8 and -9 and neutrophil-specific human neutrophil lipocalin are all increased in tobacco smokers with subclinical emphysema 42. In addition, the intracellular level of MMP-8 tends to be higher in patients with severe asthma than in those with mild-to-moderate asthma 86. Furthermore, the local concentration or activity of MMP-9 is increased in patients with asthma after allergen challenge, and MMP-9 mediates allergen-induced nonspecific hyperresponsiveness in sensitised mouse airways in vivo; these observations are fully in line with this particular proteinase having a functional impact 84, 87–89.
Reactive oxygen species
The production of cytotoxic reactive oxygen species by neutrophils is increased in patients with inflammatory lung diseases 90–93. This is of interest as there is experimental evidence in mammals that reactive oxygen species can induce the transcription of the mRNA encoding neutrophil-recruiting cytokines, such as the functionally important neutrophil chemoattractant IL-8, leading to subsequent release of IL-8 and more neutrophil recruitment 94, 95. Hypothetically, these events might contribute to altered lung function, since reactive oxygen species contribute to nonspecific bronchial hyperresponsiveness and this phenomenon may involve neutrophils in the lungs in vivo, as indicated by studies in cats, dogs and guinea pigs 64, 96–99. Conditioned medium from stimulated human neutrophils also increases the reactivity of human bronchial smooth muscle in vitro, even though it is unclear whether or not reactive oxygen species can account for such transferable effects, given their short half-lives 100.
Neutrophil-accumulating factors
Interestingly, neutrophils can produce and release the neutrophil-accumulating compounds leukotriene B4, tumour necrosis factor (TNF)-α and IL-8 38, 101–109. Thus, neutrophils themselves have the capacity to recruit even more neutrophils; this mechanism probably normally serves to mobilise large numbers of neutrophils to sites of infection. TNF-α probably also causes additional neutrophil recruitment via the stimulation of IL-8 release from bronchial epithelial cells 94, 110, 111. In addition, there is evidence that IL-8 prolongs neutrophil survival, by delaying cell death through apoptosis 112, 113. These events may have a functional impact, since there are data compatible with TNF-α and IL-8 causing bronchial hyperresponsiveness in guinea pig and mouse airways in vivo 95, 114, 115. There is also evidence that the bronchoalveolar concentration of IL-8 correlates with the degree of nonspecific bronchial hyperreactivity in mild asthma 34.
Summary
In summary, there is now convincing evidence that neutrophils contribute to bronchoconstriction, nonspecific bronchial hyperreactivity, hypersecretion in glands and lung tissue destruction. For these reasons, the endogenous mechanisms orchestrating the accumulation and activation of neutrophils constitute potential targets for novel pharmacotherapy against lung disease associated with excessive neutrophil accumulation.
ORCHESTRATION OF NEUTROPHIL MOBILISATION IN THE LUNGS
Despite recent advances in chemokine and eicosanoid biology, current understanding of the orchestration of neutrophil mobilisation remains weak; this is particularly true for the sustained mobilisation of neutrophils in chronic lung disease 23.
Cellular sources of interleukin-8
A number of different cell types in the bronchial wall, bronchoalveolar space and post-capillary venule can release potent chemoattractant signals to neutrophils. Thus, bronchial epithelial cells, bronchial smooth muscle cells, fibroblasts, monocyte-derived cells, neutrophils and even eosinophils bear the potential of contributing to neutrophil recruitment by releasing IL-8 and other CXC chemokines in diseases such as asthma and chronic bronchitis 5, 94, 110, 111, 116–120.
Effect of glucocorticoids on interleukin-8
It remains a paradox that many experimental studies indicate that glucocorticoids inhibit IL-8 production in bronchial epithelial cells, bronchial smooth-muscle cells, fibroblasts, monocytes and venous endothelial cells, whereas these normally anti-inflammatory compounds do not inhibit the accumulation and activity of airway neutrophils in patients with COPD or very severe asthma 31, 35, 78, 121–125. In line with this lack of effect on neutrophil mobilisation in certain patients, glucocorticoids do not inhibit the neutrophil chemoattractant IL-8 in severe asthma or COPD 34, 78. This observation, together with the proven ability of glucocorticoids to directly increase neutrophil survival, probably contributes to the weak effect of glucocorticoids on neutrophil mobilisation in COPD and severe asthma 126, 127. In light of these facts, it appears feasible that mechanisms other than the glucocorticoid-sensitive ones are important in orchestrating the local accumulation and activation of neutrophils through various mobilising factors in COPD and severe asthma.
T-lymphocytes and neutrophil accumulation
T-lymphocytes and eosinophil mobilisation
It has been recognised since the early 1970s that T-lymphocytes can orchestrate the sustained local accumulation and activation of eosinophils in the airways 128, through soluble factors subsequently identified as T-helper cell (Th) type 2 cytokines, including IL-3 and IL-5, as well as the growth factor GM-CSF 129–131. Strangely, the contribution of T-lymphocytes to the corresponding mobilisation of neutrophils has not received as much attention in the literature.
T-lymphocytes and neutrophil mobilisation
The acute and sustained phases of neutrophil mobilisation are key components of innate immunity contributing to host defence 18–23. The discovery of the endotoxin recognition system in a number of mammalian species, a system that potently and rapidly recruits and activates neutrophils, has underlined the potential importance of neutrophil-mobilising mechanisms in particular 18–23. Interestingly, there is now growing evidence that T-lymphocytes are involved in orchestrating the sustained mobilisation of neutrophils.
In airway tissue from patients with newly diagnosed asthma, the number of lymphocytes, eosinophils and neutrophils are increased 132. Specific blockade of lymphocytes, with an antibody directed against either CD4 or the IL-2 receptor, prevents allergen-induced recruitment of eosinophils and neutrophils in the bronchoalveolar space of sensitised mice and rats in vivo 133, 134. Importantly, in the airway tissue of certain patients with COPD, there is accumulation of both CD4+ and CD8+ lymphocytes, which is associated with accumulation of neutrophils 135, 136. Remarkably, there may even also be an association between the accumulation of CD8+ lymphocytes and neutrophils in the nasal mucosa of patients with COPD 58. Furthermore, exposure to cigarette smoke causes accumulation of CD4+ lymphocytes and neutrophils in the bronchoalveolar space of guinea pigs in vivo 137. There is also accumulation of CD4+ lymphocytes and neutrophils in airway tissue from patients with bronchiectasis 36. The accumulation of CD4+ lymphocytes is even associated with functional changes in the lungs; the local presence of CD4+ lymphocytes is related to nonspecific bronchial hyperreactivity in humans, mice and rats in vivo, even though it is not known whether this phenomenon is linked to the orchestration of neutrophils or eosinophils 129, 130, 138–140. It is of special interest that the mediator mechanisms linking CD4+ or CD8+ lymphocytes to the accumulation of neutrophils have been unclear; IL-17A now emerges as a candidate for linking activated T-lymphocytes to sustained local accumulation of neutrophils in the lungs.
THE INTERLEUKIN-17 FAMILY
Shared molecular characteristics
At present, the IL-17 family of cytokines comprises six unique homodimeric glycoproteins that share a high degree of structural homology (table 2⇓), including disulphide linkage, a relatively similar C-terminal amino acid sequence and cysteine knot folds when crystallised as monomers (fig. 1⇓) 141–143. The molecular size of the homodimeric proteins ranges 35–52 kDa, their monomers consist of ∼150–200 amino acids and their chromosomal gene localisation is widely distributed (table 2⇓).
Generic structure of monomer of the interleukin-17 family of homodimeric cytokines. The typical canonical cysteine knot fold of β-strands (▓) 1–4 is shown, including two disulphide linkages (horizontal bars between cysteine residues (╡)). N: N-terminus; C: C-terminus. (Modified from 141.)
Molecular characteristics of human interleukin-17 family of cytokines
Interleukin-17A
IL-17A (previously named IL-17 or cytotoxic T-lymphocyte-associated serine esterase-8) constitutes the prototype member of the IL-17 family of cytokines 1, 2. This particular glycoprotein consists of 155 amino acids and the molecular mass of the homodimer is 30–35 kDa 1, 2. Interestingly, mouse and rat IL-17A both display structural homology with human IL-17A; the glycosylation site appears to be highly conserved, a fact compatible with IL-17A playing an important role in the mammalian immune system 1, 2, 142.
Interleukin-17B–F
Among the additional members of the IL-17 family, IL-17F probably constitutes the one most similar to IL-17A, with 50% sequence homology over its 163 amino acids 141, 151. In contrast, IL-17B, -C, -D and -E (now renamed IL-25) all display variation in their biological profiles, in terms of both cellular sources and action 141, 143, 145–151. In line with the substantial variability among their biological profiles, their amino acid sequence homology with IL-17A is as low as 16% 145. At present, knowledge regarding the various roles of IL-17B–F in innate immunity or lung disease in humans is very limited. For this reason, this article focuses mainly on IL-17A.
CELLULAR SOURCES OF IL-17A
T-lymphocytes
Importantly, T-lymphocytes of the CD4+ and CD8+ subset that have been harvested from the spleen or blood of humans, mice or rats, most probably constituting memory (CD45RO) cells, produce and release IL-17A upon activation in vitro 2, 4, 142, 152.
Of specific interest for the potential role of IL-17A in the lungs, it was recently demonstrated that activated CD3+ lymphocytes, isolated from mouse lung tissue, release the free soluble form of IL-17A in vitro 4. As indicated for synovial CD4+ T-lymphocytes from patients with rheumatoid arthritis, the Th0 and Th1 subsets, but not the Th2 subset, produce IL-17A in vitro 153. However, there is also evidence from mice spleen CD4+ T-lymphocytes in vitro, showing that IL-17A can be produced in parallel with GM-CSF as well as TNF-α, but not with the classic Th1 and Th2 cytokines, interferon (IFN)-γ and IL-4, respectively 154. These observations question the concept that IL-17A production follows a typical Th1 or Th2 pathway. Additional in vivo evidence in favour of T-lymphocytes constituting an important source of IL-17A was recently published; separate systemic depletion of the CD4+ and CD8+ subsets of lymphocytes, respectively, results in a decrease in the concentration of IL-17A in the bronchoalveolar space of mice exposed to Gram-negative bacteria in vivo 155. Interestingly, there is now evidence from the human bronchoalveolar space favouring the idea that substantial amounts of IL-17A are not released under physiological conditions 156.
There is very little knowledge about the promoter and crucial transcription factors for IL-17A in lung lymphocytes. However, there is in vitro data on human blood T-lymphocytes of the CD8+ subset, indicating a cyclic AMP-sensitive mechanism and involvement of phosphokinase A in the transcription of IL-17A mRNA 157. There has also been a recent study on genetically engineered mice, indicating that the transcription factor signal transducer and activator of transcription-4 is involved in production of IL-17A, during formation of peritoneal abscesses after abdominal Gram-negative infection in vivo 158.
There is now evidence from mouse cells in vitro that the release of IL-17A in response to endotoxin from Gram-negative bacteria requires the presence of macrophage-like cells 4. As judged by another recent study, it seems as though the IL-17A response to Toll-like receptor-4 stimulation by live Gram-negative bacteria is triggered by IL-23, a recently described cytokine that shares a subunit with IL-12, and, just like IL-12, is produced by macrophage-like cells 155. This IL-23-mediated phenomenon does not appear to require cell-to-cell contact, and puts IL-23 forward as a key upstream regulator of IL-17A 155.
Granulocytes
Two studies have claimed that granulocytes produce IL-17A 6, 13. Thus immunoreactivity for intracellular IL-17A protein was detected in eosinophils harvested from the bronchoalveolar fluid, induced sputum and peripheral blood of patients with asthma 6. Utilising in situ hybridisation, IL-17A mRNA was present in the same eosinophils 6. The evidence promoting neutrophils as a source of IL-17A also includes detectable concentrations of IL-17A protein in bronchoalveolar fluid from endotoxin-exposed mice claiming to be lacking T-lymphocytes 13. There is also evidence of IL-17A mRNA in bronchoalveolar neutrophils, after stimulation with endotoxin or the neutrophilic cytokine IL-15 in vitro 13. It is worthy of note, though, that the cultured neutrophils were dead at the time that the extracellular IL-17A protein was detected, and that the data were referred to but not shown in the publication. Thus, it remains to be proven that live human eosinophils or neutrophils can release free soluble IL-17A upon activation in vitro.
RECEPTORS IN LUNGS
The general understanding of the receptor biology of the IL-17 family of cytokines is weak and this is also true for the lungs. At present, the most solid information relates to two subtypes of IL-17 receptors; these are the IL-17 receptor (IL-17R) and the IL-17B receptor (IL-17BR), the latter subsequently renamed IL-17 receptor homologue 1 (IL-17Rh1) 2, 141, 142, 145, 159–161.
Interleukin-17 receptor
In contrast to its protein ligands, the structure of the human variant of IL-17R displays a relatively common structure; it constitutes a type I membrane protein containing a 293-amino acid extracellular domain, a 21-amino acid transmembrane domain and a 525-amino acid cytoplasmic tail, totalling 739 amino acids 159. Interestingly, and also in contrast to its protein ligands, the cellular distribution of human IL-17 mRNA is very broad 159, 161. Thus, cells bearing the IL-17R include epithelial cells, fibroblasts, B- and T-lymphocytes, and myelomonocytic cells. In line with these findings, the organ distribution of the mRNA encoding mouse and rat IL-17R is also broad; this mRNA is detected in lungs, kidneys, liver and spleen 159. Mouse and rat cells, such as fibroblasts, epithelial cells and various cells related to T-lymphocytes, also express IL-17R mRNA 159. As revealed indirectly by immunoreactivity, IL-17R is also present in peripheral blood T-lymphocytes and vascular endothelial cells from humans 159, 161. It is of principal immunological interest that many of these cells seem to be ready for an immediate response to IL-17A under physiological conditions, since they express IL-17R constitutively. However, in terms of a general understanding of the specific receptor for IL-17A, the existing data are probably incomplete. This is because IL-17A is approximately 10 times more potent in causing secondary cytokine release than it is in specifically activating IL-17R 159. In view of this, the existence of additional facilitating subunits of IL-17R seems likely 141.
Interleukin-17 receptor homologue 1
Similarly to IL-17R, IL-17Rh1 constitutes a type I transmembrane protein, but is believed to be a smaller molecule, consisting of ∼400–500 amino acids, depending on individual report 147, 150. It displays <30% amino acid sequence homology to human IL-17R 147, 150. Expression of its mRNA appears more restricted than that of IL-17R, with weak or no expression in human lungs and almost no expression in human blood leukocytes 150. As indicated by its original name (IL-17BR), IL-17Rh1 was initially believed to specifically bind IL-17B, but a recent study has indicated that it also binds IL-17E (now renamed IL-25 147, 150).
ACTION ON NEUTROPHIL-MOBILISING FACTORS IN AIRWAY EPITHELIAL CELLS
Interleukin-8 and other CXC chemokines
Several studies have now demonstrated that stimulation of human airway epithelial cells with IL-17A in vitro causes the production and release of IL-8 3, 8–10, 162, 163. This IL-8 release is functionally significant for neutrophil recruitment; conditioned medium from IL-17A-stimulated human bronchial epithelial cells causes chemotaxis of human neutrophils, and this effect is blocked when the medium is pretreated with an antibody directed against IL-8 3. It is also noteworthy that this release of IL-8 is specific to IL-17A, since pretreatment of the recombinant IL-17A protein with a specific neutralising antibody attenuates its effect.
Stimulation with IL-17A also releases additional CXC chemokines, such as growth-related oncogene (GRO)-α and granulocyte chemotactic protein (GCP)-2 in human airway epithelial cells 162, 163. The induced mRNA expression, as well as IL-8 and GRO-α release, is of a similar order of magnitude to that after stimulation with the pro-inflammatory cytokine TNF-α, even though the absolute concentration needed to produce these responses is higher for IL-17A 8, 9. At present, there are no published studies on the involvement of nuclear factor (NF)-κB in IL-17A-induced chemokine release from human lung cells. However, as indicated in a rat intestinal epithelial cell line in vitro, IL-17A induces release of the CXC chemokine cytokine-induced neutrophil chemoattractant via a NF-κB-inducing kinase, in a TNF receptor-associated factor (TRAF)-6-dependent manner 164. The fact that NF-κB is involved in the production of IL-8 in response to a wide range of pro-inflammatory stimuli other than IL-17A in airway epithelial cells also makes an involvement of this generic transcription factor likely 165–169. Mitogen-activated protein (MAP) kinases are clearly involved as mediators of IL-17A-induced release of CXC chemokines in airway epithelial cells 9, 10, 162. Specifically, p38 and extracellular signal-regulated kinase (ERK) appear to be the most important MAP kinases in this context, even though the involvement of ERK has not yet been confirmed in primary human airway epithelial cells in vitro. In certain airway epithelial cells, the release of IL-8, GRO-α and GCP-2 caused by IL-17A display low sensitivity to a glucocorticoid, even though this may be related to the specific conditions in the in vitro model 162, 163. Of functional importance, it seems as though human IL-17 per se does not cause neutrophil chemotaxis, as indicated in blood neutrophils from humans stimulated in vitro 3.
Interleukin-6
Interestingly, human airway epithelial cells cultured in vitro respond to IL-17A by releasing cytokine IL-6, a cytokine known to increase elastase release from human neutrophils in vitro 8–10, 170. However, the involvement of the particular MAP kinase p38 remains unclear, whereas the involvement of ERK has been confirmed in primary human airway epithelial cells 9, 10. The sensitivity of this IL-6 release to a glucocorticoid is not currently known.
Colony-stimulating factors
There is evidence that IL-17A stimulates the production and release of colony-stimulating factors in human airway epithelial cells. Thus, IL-17A causes the release of G-CSF as well as GM-CSF in these cells in vitro, these two colony-stimulating factors being powerful anti-apoptotic survival factors for neutrophils 15, 127, 163, 171, 172. Interestingly, this IL-17A-induced release is also of a similar order of magnitude to that obtained after stimulation with TNF-α, as is the case for CXC chemokines, but, again, a higher absolute concentration of IL-17A is needed to produce this response. Measured as gene expression in human airway epithelial cells in vitro, IL-17A also increases G-CSF levels at a similar order of magnitude as for TNF-α 163. IL-17A may also exert a functionally significant effect on the release of GM-CSF in vitro, a release that increases the survival of human neutrophils in vitro 15. At least under these in vitro conditions, this GM-CSF release is sensitive to a glucocorticoid, but the mechanisms behind this sensitivity remain unknown.
Mucin
Among the interleukins, IL-1–18, only IL-6 and -17A are capable of directly increasing levels of mRNA encoding mucin, including MUC5AC and MUC5B, as demonstrated in primary human airway epithelial cells in vitro 16. This is also true for mucin protein itself. For IL-17A, this effect is in part mediated by IL-6, and, for MUC5B specifically, the intracellular MAP kinase ERK is involved.
Functional interactions with other cytokines
It is probably pathogenically relevant that IL-17A can interact with other pro-inflammatory cytokines when causing secondary cytokine release in local lung cells. For example, co-stimulation of human airway epithelial cells with the pro-inflammatory cytokine TNF-α enhances IL-17A-induced release of two CXC chemokines in vitro 3, 164. In a similar way, co-stimulation with the classic Th1 cytokine IFN-γ also enhances IL-17A-induced IL-8 release in human airway epithelial cells 10. Somewhat remarkably, co-stimulation with the classic Th2 cytokines IL-4 and -13, respectively, also enhances the IL-8 response to IL-17A in the same epithelial cells 10. In addition, the release of G-CSF and GM-CSF are enhanced by co-stimulation with TNF-α and IL-17A in human airway epithelial cells in vitro 15, 164. Finally, co-stimulation of human airway epithelial cells in vitro with human IL-17A plus IFN-γ markedly enhances intercellular adhesion molecule-1 levels, and this is also the case for co-stimulation with IL-4 and -13, respectively 10. Taken together, these observations are compatible with IL-17A exerting synergistic effects in Th1 as well as Th2 settings.
ACTION ON NEUTROPHIL-MOBILISING FACTORS IN NON-EPITHELIAL LUNG CELLS
Several studies now indicate that IL-17A increases the release of neutrophil-mobilising factors in local lung cells other than epithelial cells. Of particular interest, human venous endothelial cells, cells that constitute a crucial barrier for the extravasation of neutrophils, release IL-8 in response to IL-17A in vitro. This release is sensitive to a glucocorticoid, although once again, the molecular mechanisms remain unknown 3. Of additional interest, primary human lung fibroblasts, cells that possess the potential to communicate with neutrophils in local tissue, also respond to IL-17A by producing and releasing IL-6 and IL-8 in vitro 6. Similarly, in lung fibroblasts from mice, IL-17A can induce the release of the mouse CXC chemokines macrophage inflammatory protein (MIP)-2 and keratinocyte cytokine 7. By analogy, IL-17A also stimulates the release of IL-6 in embryonic lung fibroblasts 8. Again, this response to IL-17A seems to be sensitive to a glucocorticoid, even though the molecular mechanisms remain largely unknown 6. As indicated for mouse embryonic fibroblasts in vitro, the IL-17A-induced transcription and release of IL-6 is mediated via NF-κB and requires TRAF-6, but it is not known whether these mechanisms are involved in setting the sensitivity to a glucocorticoid 173. There is now also a published study on isolated primary airway smooth muscle cells from patients with bronchiolitis obliterans, which claims that IL-17A stimulates IL-8 release and that this response is not sensitive to a glucocorticoid 5. In the same type of cells, IL-17A also induces the release of 8-isoprostane, a metabolite from the peroxidation of arachidonic acid that is regarded as a marker of oxidative stress 174. Unfortunately, there are no studies on neutrophil accumulation or activity evaluating the functional significance of these in vitro findings.
At present, no thorough molecular characterisation of the mechanisms determining sensitivity to glucocorticoids of the effects of IL-17A in non-epithelial or epithelial lung cells has been published. Such a characterisation is well warranted. Data from chondrocytes suggest that IL-17A exerts its effect on secondary mediator production in its target cells via certain MAP kinases, concomitantly with the transcription factor NF-κB 170. In these chondrocytes, a glucocorticoid blunted the activation of MAP kinases, but, again, the molecular basis of this repression remains unknown 175.
ACCUMULATION OF NEUTROPHILS IN THE LUNGS
In contrast to its lack of a direct effect on neutrophil chemotaxis in vitro, IL-17A from mice, rats and humans causes substantial accumulation of neutrophils when administered locally into the airways of mice or rats in vivo 3, 14, 17, 155, 175. Interestingly, in the very first experiments in rats, conducted at a time when no rat or mouse IL-17A was commercially available, human IL-17A was administered intratracheally, and this in vivo response to human IL-17A was attenuated when the human IL-17A was pretreated with a neutralising antibody directed against hIL-17 3. This argues that, just like the IL-8 response in vitro, the in vivo response to human IL-17A is specific 3. Systemic pretreatment with a glucocorticoid attenuates this effect of IL-17A on neutrophil accumulation in the rat model, even though the molecular mechanisms remain unknown.
Local stimulation with IL-17A increases the concentration of the CXC chemokine MIP-2 in the bronchoalveolar space of rats in vivo 3. It also appears as though the IL-17A-induced release of keratinocyte cytokine, another CXC chemokine, and IL-6, respectively, also play a role in neutrophil accumulation in mouse lungs 13, 155. Interestingly, GM-CSF may also be involved in this type of neutrophil accumulation, even though normally considered to be mainly a colony-stimulating factor 15. Endogenous tachykinins enhance the referred IL-17A-induced neutrophil accumulation via neurokinin-1 receptors in rat lungs, drawing attention to a putative link to neurogenic inflammation, at least in this particular species 176.
ACTIVITY OF NEUTROPHILS IN THE LUNGS
There is evidence supporting the idea that IL-17A activates accumulated neutrophils in the bronchoalveolar space; local administration of recombinant human IL-17A causes an increase in neutrophil elastase and MPO activity in the bronchoalveolar space of rats in vivo 14. However, no corresponding effect is observed in rat neutrophils from blood after stimulation with human IL-17A in vitro, and, therefore, if a true activation effect exists, then this effect is probably mediated mainly through indirect mechanisms. No increased elastase or MPO activity is observed in the bronchoalveolar space of rats in vivo after local stimulation with recombinant rat IL-1β, using a dose equally as effective as IL-17A in terms of neutrophil accumulation 14. In spite of this, local co-stimulation with rat IL-1β plus human IL-17 displays substantially more elastase and MPO activity compared to stimulation with human IL-17A alone, thus revealing a true synergistic effect. It remains unclear whether the activating effect of human IL-17A on neutrophil elastase and MPO activity is a selective one; species heterology may be an issue for these particular findings. This would be consistent with the recent observation that local administration of recombinant mouse IL-17A increases the concentration of MMP-9 and the corresponding gelatinase activity in the bronchoalveolar space of mice in vivo, even though this increase does not cause any more MMP-9 or gelatinase activity per neutrophil 17. Thus, even though the precise mechanisms remain unclear, it seems as though the net proteolytic load in the bronchoalveolar space is increased after local stimulation with IL-17A 14, 17.
INTERLEUKIN-17A AND HOST DEFENCE IN THE LUNGS
There is a growing body of evidence, from mice in particular, in favour of IL-17A signalling playing a central role in host defence within the lungs. Thus, mice lacking IL-17R die due to lung infection caused by Klebsiella pneumoniae more frequently than do wild-type mice 11. Interestingly, the increased death rate is paralleled by weakened mobilisation of neutrophils, as well as weakened clearance of bacteria locally 177. In addition, the local increase in IL-17A concentration parallels bacterial growth in this mouse model of Gram-negative lung infection 177. In line with a specific role in host defence, local administration of endotoxin from Escherichia coli, another Gram-negative bacteria, requires the presence of endogenous IL-17A, not for the early phase, but for the sustained phase of neutrophil accumulation in the bronchoalveolar space of mice in vivo 4, 13. It is also clear that local administration of this endotoxin increases the relative amount of IL-17A mRNA in the lungs and the concentration of IL-17A in the bronchoalveolar space of mice in vivo 4, 178. In addition, adenovirus-mediated overexpression of IL-17A stimulates granulopoiesis in mice in vivo in a stem cell factor- and G-CSF-dependent manner 179, 180. All of these findings are compatible with the T-lymphocyte cytokine IL-17A constituting a crucial factor in the orchestration of the neutrophil component in host defence. However, importantly, corresponding evidence from studies in human lungs is still lacking.
INTERLEUKIN-17A IN LUNG DISEASE
The causative role of IL-17A in human lung disease is very much unknown. A substantial increase in the concentration of free soluble IL-17 within the bronchoalveolar space has been demonstrated in healthy human volunteers, after exposure to organic dust in a swine confinement area 156. This local increase in IL-17A parallels a substantial local accumulation of neutrophils and a more moderate accumulation of lymphocytes. However, at present, it is not known how this increase in IL-17A relates to the increased morbidity in lung disease observed among swine farmers with regular exposure to organic dust 181.
Certain patients with mild asthma may display a modest increase in the concentration of free soluble IL-17A within the bronchoalveolar space 6. This IL-17A concentration increase is associated with a more pronounced increase in indirect immunoreactivity for intracellular IL-17A in inflammatory cells from the bronchoalveolar space. However, one recent study on induced sputum claims that there is no major difference in the concentration of IL-17A in patients with moderate asthma and healthy control subjects 182. Even though the number of included subjects was limited, the data from the very same study suggest that the local concentration of IL-17A is higher in asthma than in moderate COPD. In this particular study, a correlation between the local concentration of IL-17A and the degree of nonspecific bronchial hyperreactivity was reported but this report was based upon an analysis of pooled material originating from healthy control subjects, as well as from patients with asthma or COPD. The study included no analysis of the association of local IL-17A with inflammatory cell subtypes. At present, there are no further studies on IL-17A in human lung disease, even though there is a recently published clinical study showing increased expression of IL-17A in the nasal mucosa of patient with nasal polyps, compatible with IL-17A being linked to atopic airway disease 183.
Potentially important clues regarding the potential role of IL-17A in lung disease are also provided by studies on sensitised mice. It has now been shown that allergen challenge is followed by transcription of IL-17A in mouse lungs 12. The indicated de novo synthesis of endogenous IL-17A also seems to parallel allergen-induced neutrophil accumulation in the bronchoalveolar space 12. Furthermore, inhibition of endogenous IL-17A even increases allergen-induced eosinophil accumulation and elevates levels of the Th2 cytokine IL-5 in the same compartment, suggesting that IL-17A may have an impact on the balance between eosinophilic and neutrophilic inflammation in mouse lungs. Another study on sensitised mice has provided data compatible with endogenous IL-17A also mediating allergen-induced nonspecific bronchial hyperresponsiveness, possibly by contributing to the activation of T-lymphocytes 184.
CONCLUSIONS AND FUTURE QUESTIONS
Recent studies promote IL-17A as a candidate cytokine for linking the activation of T-lymphocytes to the sustained accumulation and activity of neutrophils locally in the lungs. IL-17A seems to be most important not for the early phase but for the sustained phase of neutrophil mobilisation in host defence in the lungs. It is, therefore, possible that certain T-lymphocytes play both complementary and supplementary roles to well-known neutrophil-mobilising cells, such as the bronchial epithelium and bronchoalveolar macrophages. IL-17A may thus be uniquely positioned in the interface between acquired and innate immunity, as a reinforcing signal. It remains an open question as to whether inflammatory cells other than subsets of T-lymphocytes also release IL-17A in the lungs. Regardless of its cellular origin, IL-17A appears to exert its effects on the sustained mobilisation of neutrophils primarily via an induced release of neutrophil-mobilising cytokines from local cells in the lungs. Whether IL-17A is mainly important for host defence, or whether this molecule also plays a pathogenic role, in particular in chronic lung disease associated with excessive neutrophil activity, is not yet certain.
Future studies should further evaluate the therapeutic utility of drugs targeting interleukin-17A and its receptor(s), a strategy that has now also been discussed for organs other than the lungs 185–187.
- Received March 16, 2004.
- Accepted August 5, 2004.
- © ERS Journals Ltd
References
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- Article
- Abstract
- NEUTROPHILS AND INNATE IMMUNITY
- ACCUMULATION OF NEUTROPHILS IN LUNG DISEASE
- ACCUMULATION OF NEUTROPHILS AND LUNG FUNCTION
- NEUTROPHIL PRODUCTION OF COMPOUNDS WITH PATHOGENIC POTENTIAL
- ORCHESTRATION OF NEUTROPHIL MOBILISATION IN THE LUNGS
- T-lymphocytes and neutrophil accumulation
- THE INTERLEUKIN-17 FAMILY
- CELLULAR SOURCES OF IL-17A
- RECEPTORS IN LUNGS
- ACTION ON NEUTROPHIL-MOBILISING FACTORS IN AIRWAY EPITHELIAL CELLS
- ACTION ON NEUTROPHIL-MOBILISING FACTORS IN NON-EPITHELIAL LUNG CELLS
- ACCUMULATION OF NEUTROPHILS IN THE LUNGS
- ACTIVITY OF NEUTROPHILS IN THE LUNGS
- INTERLEUKIN-17A AND HOST DEFENCE IN THE LUNGS
- INTERLEUKIN-17A IN LUNG DISEASE
- CONCLUSIONS AND FUTURE QUESTIONS
- References
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