Background
Monocytes can be recruited from the blood into the tissues, whereupon differentiation into macrophages may occur [
1]. There are also tissue resident macrophages that replenish cell numbers by replication [
2]. A recent study demonstrated the presence of phenotypically different mononuclear phagocyte cell types in healthy human lungs that either originate from the lungs (pulmonary dendritic cells and alveolar macrophages) or from blood monocytes (monocyte derived cells and tissue monocyte/macrophages) [
3].
There are increased numbers of macrophages in the lungs of chronic obstructive pulmonary disease (COPD) patients [
4]; these cells are involved in host defence, airway remodelling and parenchymal destruction [
5]. It has been suggested that increased lung macrophage numbers in COPD are due to increased recruitment of blood monocytes [
5,
6]. Alternatively, cigarette smoke exposure induces the expression of anti-apoptotic genes in macrophages [
7], and increased expression of anti-apoptotic proteins has been observed in COPD macrophages [
8], suggesting that delayed apoptosis is a possible cause of macrophage accumulation in COPD. Furthermore, alveolar macrophages expressing the proliferation marker Ki67 have been observed in patients with interstitial lung disease [
9], but whether increased macrophage accumulation in COPD occurs by self-renewal is not understood.
Costa et al reported increased migration of COPD peripheral blood mononuclear cells towards C-X-C motif chemokine receptor 3 (CXCR3) and C-C motif chemokine receptor 5 (CCR5) ligands using single chemokines for migration experiments [
6]. Such experiments, however do not reflect the complex mixture of chemoattractants present in the lungs [
10‐
15]. Physiologically relevant complex supernatants, such as those obtained from induced sputum could be used to further investigate the migratory ability of COPD monocytes.
CCR5 is the receptor for the monocyte chemoattractant C-C motif chemokine ligand 3 (CCL3) [
16]. Studies using induced sputum and bronchoalveolar lavage have shown that CCR5 ligand levels are increased in the lungs of COPD patients, suggesting a role for CCR5 signalling in the recruitment of monocytes into COPD lungs [
12,
13,
16,
17]. Peripheral blood monocytes can be classified into 3 subtypes according to their expression of CD14 (LPS receptor) and CD16 (FcγRIII receptor): CD14
++CD16
- (‘Classical’), CD14
+CD16
+ (‘Intermediate’) and CD14
-CD16
++ (‘Non-Classical’) [
1]. Increased numbers of pro-inflammatory CD14
+CD16
+ monocytes are found in chronic inflammatory disease states such as rheumatoid arthritis [
18]. Furthermore, CD14
+CD16
+ cells have the greatest surface expression of CCR5 [
1,
19]. Monocyte subsets in COPD, and their expression of CCR5, have not been previously reported.
CCR5 expression is upregulated by interleukin-6 (IL-6) [
20], a cytokine which trans-signals through a soluble receptor sIL-6R [
21]. Plasma IL-6 levels are increased in a subset of stable COPD patients [
22] and during COPD exacerbations [
23]. The systemic levels of sIL-6R have not been investigated in COPD; increased systemic IL-6/sIL-6R signalling in COPD could upregulate blood monocyte CCR5 expression, thereby promoting monocyte recruitment into the lungs.
We have investigated COPD blood monocyte recruitment with two major objectives in mind. Firstly, to characterise changes in the CCL3-CCR5 axis that could facilitate monocyte recruitment in COPD; we studied CCR5 expression on peripheral blood monocytes and plasma sIL-6R levels in COPD patients compared to controls. Secondly, to further investigate the hypothesis that monocyte recruitment from the blood is increased in COPD; we studied COPD monocyte migration towards sputum supernatants and performed lung immunohistochemistry studies to evaluate monocyte migration from the pulmonary blood vessels of COPD patients compared to controls. We also performed immunohistochemistry studies to investigate an alternative mechanisms of pulmonary macrophage accumulation in COPD; namely increased replication and supressed apoptosis.
Discussion
We observed increased CCR5 expression on COPD blood monocytes. Increased plasma levels of sIL-6R may play a role in this observation, as IL-6 with sIL-6R upregulated CCR5 gene expression in monocytes. However, this increase in COPD monocyte CCR5 expression did not confer a greater migratory ability; chemotaxis experiments showed impairment of the migratory ability of COPD peripheral blood monocytes. This impaired migration was confirmed by examining monocyte margination from blood vessels in the lungs. We have therefore found no evidence to support the theory that increased monocyte recruitment is responsible for increased lung macrophage accumulation in COPD.
We investigated alternative mechanisms that could contribute to lung macrophage accumulation in COPD. There were low levels of alveolar macrophage self-renewal in COPD, suggesting that this mechanism does not contribute to the macrophage accumulation in COPD. Current smokers, with and without COPD, had increased expression of the anti-apoptotic marker BCL-2 in alveolar macrophages. These findings suggest a mechanism by which macrophage accumulation can occur in smokers and COPD patients.
The mechanisms for impaired COPD monocyte migration may be related to systemic oxidative stress, which is increased in S [
29] and COPD [
29,
30]. It is known that monocytes from S display migratory impairment [
31]; this phenomenon can be induced in HNS monocytes after exposure to radical and non-radical oxidants [
31]. We observed a trend toward significant reduction in monocyte migration when comparing S and HNS. Inflammatory cell chemotaxis may diminish with increasing age [
32], however a subgroup analysis of our data in HNS monocytes failed to show such an association.
A recent study showed that lymphocytes and monocytes interact to facilitate peripheral blood mononuclear cell (PBMC) chemotaxis towards CXCR3 and CCR5 ligands, but that isolated COPD monocytes had similar chemotaxis ability to controls [
6]. We used a more complex chemotactic system with induced sputum supernatant and isolated monocytes. We have previously shown that CCL3 (a ligand for CCR5) levels are increased in COPD sputum, and that CCL3 and CCR5 blockade reduces monocyte chemotaxis to COPD sputum supernatant [
16]; we now demonstrate that exposure to rhCCL3, induces migration of COPD CD14
+ monocytes. We also observed increased CCR5 expression on CD14
++CD16
- and CD14
+CD16
+ COPD blood monocytes, so it was perhaps surprising that COPD monocytes displayed impaired chemotaxis. It was therefore important that we evaluated monocyte migration into the lungs by a different method to further investigate this observation.
C-X3-C motif chemokine receptor 1 (CX
3CR1) is a widely used monocyte/macrophage marker [
1,
33]. We found reduced margination of CX
3CR1
+ cells in COPD compared to NS. The vascular endothelium of pulmonary vessels in S and COPD expresses increased levels of adhesion molecules such as E-selectin, P selectin, ICAM-1, ICAM-2 and VCAM-1 [
34]. It is therefore unlikely that the reduced margination observed in COPD resulted from attenuated endothelial adhesion molecule expression. The reduced margination was observed for both CX
3CR1
+CD14
+ and CX
3CR1
+CD16
+ cells in COPD patients.
Attenuated monocyte migration ability in COPD raises questions regarding the mechanisms of increased lung macrophage numbers in COPD patients. Desch et al. recently described a “tissue monocyte” population in healthy human lungs that resembles monocytes, but expresses cell surface markers found in alveolar macrophages [
3]. Populations described as “monocyte derived cells” were also identified, which are thought to be monocytes that change phenotype and acquire new cell surface markers including CD206 when recruited from the blood into the lungs, as previously reported in mice [
35]. Our results support the concept that monocytes can be recruited into the lungs, as we observed marginated monocytes in control lung samples. However, monocyte margination was reduced in COPD patients, indicating that increased lung macrophage numbers in COPD are not simply due to excessive blood monocyte recruitment.
Murine studies have shown that lung macrophages can be replenished by self-renewing, locally-derived progenitor cells [
2,
36]. Human studies have also shown that resident lung mononuclear phagocyte populations can express the cell-cycle maintaining protein Ki67, suggesting that these cells are engaged in self-renewal [
37,
38]. We observed low levels of Ki67 expression amongst alveolar macrophages, suggesting that a very limited proportion of these cells were actively undergoing self-renewal. Simian studies have suggested that the turnover of these cells is indeed very low whereas the turnover of interstitial lung macrophages is high [
39].
It has been reported that COPD lung macrophages express increased levels of a protein known as ‘Apoptosis inhibitor of macrophage (AIM)’ which is associated with delayed apoptosis [
8]. A different study reported that alveolar macrophages from smokers displayed higher levels of anti-apoptotic proteins including Bcl-
xL [
7]. These previous studies support the hypothesis that delayed apoptosis, caused by cigarette smoking, contributes to macrophage accumulation in COPD. We observed that the anti-apoptotic protein BCL-2 was only present in current smokers, with and without COPD. Our findings support a role for current smoking in prolonging alveolar macrophage lifespan. We speculate that the increased alveolar macrophage numbers due to active smoking does not return to normal after smoking cessation, as the years of chronic cigarette smoking causing delayed apoptosis have permanently altered the homeostasis of lung macrophage numbers. We also note that Kojima et al found increased AIM expression in COPD alveolar macrophages compared to both smoking and non-smoking controls, implicating this particular anti-apoptotic protein in COPD specific mechanisms [
8]. Overall, these previous findings and our current observations indicate mechanisms of delayed apoptosis that can occur in macrophages from current smokers or COPD patients.
In keeping with previously published studies, we observed increased plasma IL-6 levels in stable COPD patients [
22]. We also observed significantly increased plasma sIL-6R levels in COPD patients compared to S. IL-6 signals through either membrane-bound IL-6R or sIL-6R [
21]. Increased sIL-6R levels may amplify the effects of IL-6 [
40]. CCR5 gene expression in microglial cells is upregulated following culture with IL-6 [
20]; in the same study IL-6 stimulation caused a numerical, but not statistically significant, increase in CCR5 gene expression in healthy blood monocytes [
20]. We also observed a small increase in CCR5 expression with IL-6 alone, but significant induction was achieved when sIL-6R was also present, suggesting an important role for IL-6 trans-signalling in the regulation of CCR5 expression. An alternative mechanism for CCR5 upregulation in monocytes is the effects of reactive oxygen species exposure, which can upregulate CCR5 expression [
41,
42].
Increased sIL-6R levels may promote inflammatory activity in COPD by enhancing the effects of IL-6 through trans-signalling. IL-6 is involved in the polarization of naïve CD4
+ T lymphocytes towards the pro-inflammatory Th17 effector phenotype [
43]; furthermore, IL-6 suppresses apoptosis of both innate and adaptive immune cells resulting in their persistence at foci of inflammation [
44,
45].
CD14
+CD16
+ monocytes are potent secretors of IL-1, IL-6 and TNF-α [
1], and expanded CD14
+CD16
+ monocyte populations are found in inflammatory disease states including atherosclerosis [
46], obesity [
47] and rheumatoid arthritis [
48]. We did not observe any change in monocyte subsets in COPD patients compared to controls. Furthermore, there was no change in cellular subpopulations during exacerbations, indicating no dysregulation of CD14
+CD16
+ cells in COPD.
We elected to use sputum for chemotaxis experiments as there are increased levels of monocyte chemoattractants in the sputum supernatants of COPD patients compared to controls [
14,
16]. Furthermore, the total number of macrophages in sputum is increased in COPD patients [
14,
16]. Bronchoalveolar lavage supernatants are an alternative for chemotaxis experiments, representing a different lung compartment where macrophages numbers are increased. However, this is more invasive and the installation of saline may cause excessive dilution.
Acknowledgements
We are grateful to Dr Louise Healy (UCB, Clough UK) for her assistance with the MSD® analysis of plasma samples. The COPD Assessment Test was reproduced with the permission of GlaxoSmithKline (GSK, Brentford UK). GSK owns the Intellectual Property in the COPD Assessment Test. The SGRQ was reproduced with the permission of St George’s University, London (UK). We also wish to thank Ms Antonia Banyard for her generous technical assistance with regards the flow cytometric characterization of monocytes.