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. 2006 Feb;143(2):363–372. doi: 10.1111/j.1365-2249.2006.02998.x

Alveolar macrophage function is altered in patients with lung cancer

D S Pouniotis *, M Plebanski *, V Apostolopoulos *,‡, C F McDonald †,‡
PMCID: PMC1809587  PMID: 16412062

Abstract

The alveolar macrophage (AM) is believed to be of central importance in the immune response against infection and tumour. We examined patients with lung cancer in order to evaluate the immuno-stimulatory potential of AM in lung cancer. Bronchoalveolar lavage fluid samples were obtained from patients with adenocarcinoma, squamous cell carcinoma, large cell undifferentiated lung carcinoma, small cell carcinoma and control subjects. AM were isolated and phagocytic function, flow cytometry and cytokine analysis were assessed. AM from patients with small and squamous cell carcinoma had impaired uptake in vitro of 40 nm fluorescent polystyrene beads. AM from patients with small, squamous and large cell undifferentiated carcinoma showed impaired uptake of 1000 nm fluorescent polystyrene beads. Secreted levels of TNF-α and IL-1 from AM of patients with small, squamous, and large cell undifferentiated carcinoma were decreased compared to controls. Secreted AM IL-6 levels were decreased in small and large cell undifferentiated carcinoma. AM from adenocarcinoma patients showed similar levels of IL-10, IL-6, IL-1 and TNF-α compared to controls. Phenotypic analysis demonstrated that patients with small cell carcinoma were the only group that showed a decrease in MHC class II surface expression. Surface expression of ICAM-1 and CD83 was decreased on AM from patients with large, squamous and small cell carcinoma compared to controls but not adenocarcinoma. Mannose receptor levels were only decreased on AM from patients with squamous and small cell carcinoma but not adenocarcinoma and large cell undifferentiated carcinoma. We conclude that there are type-specific alterations in uptake ability, cytokine secretion and phenotype of AM from lung cancer patients, which may result in an inability to stimulate anti-tumour immunity. The observed differences between lung cancer subgroups may explain previously reported inconsistencies in descriptions of AM characteristics in lung cancer.

Keywords: alveolar macrophage, phenotype, cytokines, lung cancer

Introduction

Lung cancer is the leading cause of cancer death among men and women in the United States and throughout the developed world. Despite better surgical techniques and developments in radiotherapy and chemotherapy, it is still the case that fewer than 15% of patients with this disease survive five years [1]. Improved understanding of tumour biology and the host response to the tumour may enable the development of novel therapeutic strategies for managing this disease.

Macrophages are mononuclear phagocytic cells that develop in a variety of tissues by maturation and differentiation from blood monocytes. Their major functions within the immune system are:

  • to regulate local inflammatory reactions by the release of pro- and anti-inflammatory cytokines;

  • to provide a primary defence mechanism via phagocytosis and respiratory burst;

  • to mediate immune responses through antigen processing and presentation.

Macrophages may exert direct cytotoxic effects on tumour cells or may produce anti-tumour effects through the release of a wide variety of cytokines [2]. The alveolar macrophage (AM) is likely to be of central importance in the lung cancer immune response, although the relationship between AM and tumour-associated macrophages in this condition is unclear. Although some studies have found tumour-associated macrophage numbers in non small cell lung carcinoma to be positively correlated with tumour regression [3], the presence of tumour-infiltrating macrophages was associated in other studies with increased microvessel counts and poorer prognosis in non small cell lung carcinoma [4]. Up-regulation of IL-8 expression by infiltrating macrophages correlates with tumour angiogenesis and reduced patient survival in non small cell lung carcinoma [4]. Indeed it has been suggested that these cells may have a tumour-promoting role through their production of angiogenic factors such as platelet-derived growth factor (PDGF) [5] and vascular endothelial growth factor (VEGF) [6]. Clearly, the role of tumour associated macrophages in lung cancer growth is complex. We have previously demonstrated that pulmonary AM from patients with cancer have reduced cytostatic activity and reduced production of IL-1 compared with those from control subjects without cancer [7,8]. In the current study we have extended our previous work by examining a larger sample of individuals with all types of primary lung cancer in order to more extensively evaluate AM functional activity and phenotype and to determine whether any alterations in immune functions of these cells are tumour-specific. As well as cytokine producing capacity, the immuno-stimulatory potential of AM from patients with various types of primary lung cancer was studied. Phenotypic analysis of surface markers important for stimulation of T cell immunity, pro- and anti-inflammatory cytokine production and uptake studies was also used to assess their function.

Materials and methods

Study participants

Bronchoalveolar lavage (BAL) fluid samples were obtained from patients with provisionally diagnosed primary lung cancer and control subjects. Patients with subsequently proven lung cancer were undergoing diagnostic bronchoscopy in all except one case. In all cases bar one, the bronchoscopy test was performed in order to confirm a suspected diagnosis of lung cancer. Thus, none of the patients (except for only one patient who had small cell carcinoma and had previously received chemotherapy) had yet received either chemotherapy or radiotherapy as treatment for their cancer. The control subjects had all presented with chronic cough or haemoptysis and normal chest radiograph, and were undergoing routine bronchoscopy to exclude endobronchial abnormality. Patient demographic details are presented in Table 1. Ethics committee approval was received from Austin Health Ethics Committee, Heidelberg VIC Australia and informed consent of all participating subjects was obtained.

Table 1.

Demographic details of lung cancer and control patients.

Groups Total number Age (years) mean ± SD Ex-smoker Current smoker Non-smoker
Control 12 64 ± 9·8 5 5 2
Adenocarcinoma 9 74·5 ± 9·02 6 2 1
Squamous cell carcinoma 6 67·7 ± 11·2 5 1 0
Small cell carcinoma 6 68·3 ± 14·8 4 2 0
Large cell carcinoma 7 61·8 ± 11·5 3 4 0
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Bronchoalveolar lavage (BAL) fluid procedure

BAL was performed at the time of diagnostic fibreoptic bronchoscopy using warm sterile 0·15 M NaCl instilled in 20 ml aliquots to a total volume of not greater than 150 ml. In patients with endobronchial lesions, the lobe lavaged was the nearest lobe which appeared macroscopically normal. Where no endobronchial abnormality was present, the lobe containing the lesion was lavaged. The endobronchial findings in all control subjects were normal, and in this group either the right middle lobe or the lingula lobe was lavaged. BAL fluid was aspirated, collected on ice and transferred to polypropylene containers in order to minimize macrophage adherence. Samples were refrigerated for a maximum of 30 min after collection before transfer to the laboratory.

AM enrichment

Total cell counts were obtained prior to AM enrichment and were not different between the groups (not shown). Every precaution was taken to prevent inadvertent activation of AM upon harvesting and adherence in order to closely mimic the AM in the original milieu. BAL fluid was washed in AIM-V media (Gibco, UK) supplemented with nystatin and AM obtained after adherence of cells (4 × 106 cells/5 ml) for 3 h at 37 °C (in AIM-V media containing nystatin). The nonadherent cells were discarded and adherent cells were removed and counted using 0·4% (w/v) trypan blue to exclude dead cells. All AM were cultured at a concentration of 1 × 106/ml in 24 well plates in AIM-V media with lipopolysaccharide (LPS; 5 µg/ml) stimulation for 18 h.

Uptake studies

AM were incubated at 1 × 106/ml in 24 well plates for 18 h with 5 µg/ml of LPS (Sigma, Australia) and carboxylated 40 nm and 1000 nm polystyrene microspheres with fluorescent phycoerythrin (PE) labels (Molecular Probes, Eugene, OR, USA). Fluorescent beads were stored at 4°C and sonicated for 15 min before use. Cells were collected and incubated on ice for 45 mins with FITC-conjugated monoclonal antibody to CD68 (Dako, Australia) in cold 2% (w/v) BSA/PBS, washed twice and analysed by flow cytometry (BD Biosciences, San Jose, CA, USA). Percentage surface expression (± standard deviation, SD) was assessed and the mean ± SD of double positive CD68/PE-fluorescent beads was determined for lung cancer and control patients.

Cytokine ELISA

Supernatants from AM incubated with LPS for 18 h were collected. ELISA plates were coated with purified human IL-1, TNF-α, IL-6 and IL-10 antibody (Pharmingen, San Diego, CA, USA) and incubated overnight at 4 °C in humidified conditions. Plates were washed five times with 0·05% (v/v) Tween 20/PBS (PBS-T) and blocked with 10% (w/v) BSA/PBS for 1 h at room temperature. 50 µl of sample supernatant in triplicates were added and left for 2 h at room temperature in humidified conditions. Plates were washed again as described above and biotin-conjugated human anti-IL-1, TNFα, IL-6 and IL-10 antibodies (Pharmingen) were added for 1 h at room temperature. After washing plates again, streptavidin HRP (SILENUS Laboratories, Melbourne, Australia) was added for 1 h and responses were detected with ABTS substrate and read at OD 405. Standard curves were prepared in PBS solution using commercially available recombinant human IL-1, TNFα, IL-6 and IL-10 (Pharmingen). Values are shown as mean pg/ml ± SD for each primary lung cancer group and control patients.

Phenotype/flow cytometry studies

The expression of CD68 (Dako, Botany, NSW, Australia), HLA-DR, HLA-DQ, ICAM-1 (CD54), CD14, mannose receptor (MR) and CD83 (Pharmingen) on AM was assessed using flow cytometry. AM were stained by incubating the cells with different combinations of antibodies directly conjugated to fluorescent probes (CD68/HLA-DR,DQ, CD68/ICAM-1, CD68/CD14, CD68/MR, CD68/CD83) for 45 min at 4 °C. 1 µg/ml of propidium iodide was added to samples just before analysis to exclude dead cells and adjust settings for autofluorescence. Purity of AM was assessed according to CD68 and HLA-DR,DQ surface expression (> 90%) and at least 5000 cells were counted in this gate. All quadrants were set up according to matched isotype control antibodies and all results are shown as percentage surface expression. Percentage surface expression (SE) was assessed and the mean SE of double positive cells is shown ± SD for lung cancer and control groups.

Statistical analysis

Assays were performed in triplicate. Mean values were compared using the two-tailed unpaired t-test. Two P-value thresholds are noted in the text; P < 0·001 to indicate a highly significant difference, and P < 0·05 to indicate a significant difference.

Results

Uptake of polystyrene particles by AM

AM from control patients were able to take up the majority of 40 nm beads after 24 h (81% ± 11%). AM from patients with squamous cell carcinoma (39% ± 20%) and small cell carcinoma (29% ± 11%) were impaired in their ability to take up 40 nm beads compared to control patients (Fig. 1a). AM from patients with adenocarcinoma (63% ± 19%) and large cell undifferentiated carcinoma (62% ± 18%) showed similar uptake of 40 nm beads to control patients in vitro. AM from control patients showed a similar ability to take up large 1000 nm beads after 24 h compared to 40 nm beads (83% ± 10%). However, the uptake of 1000 nm beads was impaired in all patients with lung cancer (P < 0·05 for adenocarcinoma, squamous and large cell carcinoma; P < 0·01 for small cell carcinoma) compared to control patients (Fig. 1b). Representative dot plots of 40 and 1000 nm fluorescent bead uptake from one patient in each study group is shown (Fig. 1c).

Fig. 1.

Fig. 1

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Uptake of different size particles by AM. AM from BAL samples from adenocarcinoma (adeno) (n = 4), small cell carcinoma (small) (n = 5), squamous cell carcinoma (squamous) (n = 4) and large cell undifferentiated carcinoma (large) (n = 6) compared to control subjects (n = 12) were incubated with (a) 40 nm or (b) 1000 nm PE-fluorescent polystyrene beads for 24 h with LPS (5 µg/ml). AM were costained with CD68 and percentage surface expression was assessed via flow cytometry. (c) Shows representative flow cytometry dot plots of one patient from each lung cancer group compared to control patients. Unpaired t-test statistical analysis was performed on cancer patients compared to noncancer patients. *P < 0·05, **P < 0·001.

Cytokine production by AM

We analysed the cytokine production of LPS activated AM from lung cancer patients compared to noncancer patients (Fig. 2). There was no detectable IL-10 in AM supernatants from patients with small, large and squamous cell carcinoma (< 10 pg/ml) (Fig. 2a). IL-10 levels were low but measurable in control patients and there was no significant difference between control (60 ± 54 pg/ml) and adenocarcinoma patients (53 ± 40 pg/ml) (Fig. 2a). Secretion of IL-1 was greatly decreased from AM of patients with small cell (22 ± 19 pg/ml, P < 0·05), large cell (6 ± 6 pg/ml, P < 0·05) and squamous cell carcinomas (19 ± 5 pg/ml, P < 0·05) but not adenocarcinoma (261 ± 122 pg/ml) compared to control patients (504 ± 265 pg/ml) (Fig. 2b). TNF-α was decreased in AM from all groups of lung cancer patients except adenocarcinoma (Fig. 2c), however, only AM from patients with small (7·4 ± 9·8 pg/ml, P < 0·05) and large cell carcinoma (8·9 ± 7·55 pg/ml, P < 0·05) showed a decrease in IL-6 secretion (Fig. 2d).

Fig. 2.

Fig. 2

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Cytokine secretion profiles of AM. AM from BAL samples from adenocarcinoma (adeno) (n = 6), small cell carcinoma (small) (n = 5), squamous cell carcinoma (squamous) (n = 6) and large cell undifferentiated carcinoma (large) (n = 7) compared to control patients (n = 12) were stimulated with LPS (5 µg/ml) for 18 h and supernatants collected for analysis by cytokine ELISA. (a) IL-10 (b) IL-1 (c) TNF-α and (d) IL-6 were tested and mean values ± SD shown. *P < 0·05, **P < 0·001 for cancer versus noncancer results.

Phenotype of AM

Phenotypic analysis of AM from patients with adenocarcinoma, small, squamous and large cell undifferentiated carcinoma compared to control patients (n = 12) was determined (Figs 3 and 4). Phenotypic expression of AM varied between cancer and control groups and between the various types of lung cancer. The most notable difference was observed on AM from small cell carcinoma patients where the level of surface expression of all markers tested, HLA-DR (MHC class II), ICAM-1, mannose receptor and CD83 was decreased (Figs 3 and 4). Within the non small cell carcinoma group there were noticeable differences between groups. AM from adenocarcinoma patients had similar levels of HLA-DR (90·5 ± 3·5%), ICAM-1 (66 ± 14·6%), CD83 (21·3 ± 11%) and mannose receptor (10·4 ± 4·8%) to control patients (Figs 3 and 4). Squamous cell carcinoma patients showed similar levels of HLA-DR (96·5 ± 2·2%) to controls but significantly decreased levels of ICAM-1 (14·2 ± 9·2%, > 4-fold), mannose receptor (1·8 ± 2·1%, > 3–5-fold) and CD83 (2·2 ± 2·44%, > 5-fold). Surface expression of HLA-DR, ICAM-1 and CD83 were moderately reduced on AM from patients with large cell undifferentiated or undifferentiated nonsmall cell lung carcinoma patients but mannose receptor was not affected. Small cell carcinoma patients showed highly significant decreases in all surface markers tested (Figs 3 and 4). CD14 expression was tested on all patient samples and no differences were noted between any of the patient groups (not shown).

Fig. 3.

Fig. 3

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MHC class II expression of AM. AM from BAL samples from adenocarcinoma (adeno) (n = 6), small cell carcinoma (small) (n = 5), squamous cell carcinoma (squamous) (n = 6) and large cell undifferentiated carcinoma (large) (n = 8) compared to control patients (n = 12) were stimulated with LPS (5 µg/ml) for 18 h and stained with monoclonal antibodies against CD68 and costained with HLA-DR,DQ (MHC class II). (a) Mean values of percentage surface expression are given ± SD. (b) Representative flow cytometry dot plots of one patient from each primary lung cancer groups compared to control patients. *P < 0·05, **P < 0·001 for cancer versus noncancer results.

Fig. 4.

Fig. 4

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Phenotypic characteristics of AM in cancer compared to noncancer patients. AM from BAL samples from adenocarcinoma (adeno) (n = 6), small cell carcinoma (small) (n = 5), squamous cell carcinoma (squamous) (n = 6) and large cell undifferentiated carcinoma (large) (n = 7) compared to control patients (n = 12) were stimulated with LPS (5 µg/ml) for 18 h and stained with monoclonal antibodies against CD68 and costained with (a) ICAM-1 (CD54), (b) mannan and (c) CD83. Mean values of percentage surface expression are given ± SD. (d) Representative flow cytometry dot plots of one patient from each primary lung cancer group compared to a control patient. *P < 0·05, **P < 0·001 for cancer v noncancer results.

Discussion

Macrophages have been implicated in the body's immune response against cancer, although they have been described as a ‘double edged sword’ because of their potential to also promote tumour progression, in part by contributing to tumour stroma formation and angiogenesis through their release of PDGF [2,5] and VEGF [6] and by increasing tumour cell IL-8 secretion [9]. In lung cancer, tumour-associated macrophage infiltration has been associated with both tumour regression and tumour progression [3,10]. The balance between tumour-promoting and tumour-suppressing effects of macrophages may depend upon the degree of activation of macrophages in the vicinity of the tumour, and upon the presence of suppressive signals produced by the tumour.

We have previously found defects in cytostatic activity and cytokine production of AM from patients with primary lung cancer compared with those from control subjects [7,8]. Others have shown AM from patients with primary lung cancer suppressed chemotaxis and this intrinsic functional defect is more pronounced on AM from the local tumour site compared to the opposite side of the lung [3]. Eifuku et al. [11] reported heterogeneous cytotoxic function of BAL AM in patients with lung cancer and suggested that the heterogeneity of response likely reflected variations in activation state of AM. Heterogeneity in cytotoxicity against the squamous cell lung cancer cell line QG56 was also noted in response to IFN-γ stimulation in this study, where AM cytotoxic potential paralleled AM IL-1 secretion. Previous reports on the functional changes of AM in patients with lung cancer demonstrated either reduced secretion of various cytokines or no change compared with controls. Tumour-associated macrophages from patients with non small cell lung cancer have been demonstrated to have reduced production of IL-1, IL-6, TNF-α, TGF-β [12] as well as reduced chemotactic activity, antibody-dependent cell mediated cytotoxicity and cytostatic activity. These results are similar to those reported by us previously [7,8]. These same AM were however, able to be induced in vitro to secrete increased amounts of these cytokine after stimulation. The current studies extended our previous observations of AM in lung cancer by firstly looking at a range of functions including phagocytosis and an extended range of cytokine production, broadening our range of cell surface markers and including larger numbers of patients in each subgroup of lung cancer to enable definition of any type-specific alterations.

AM are able to function as efficient scavengers through phagocytosis of particles, pathogens, apoptotic cells and other substances in the extracellular environment. AM also take up antigen for processing and presentation on MHC class I and II molecules to T cells [13]. AM function was assessed in the current studies by adding 40 nm and 1000 nm fluorescent latex beads to AM cultures and then determining their presence within AM at 24 h by flow cytometry. AM from patients with squamous and small cell carcinoma were impaired in their ability to take up 40 nm beads compared to control subjects while patients with squamous, small and large cell carcinoma showed an impaired ability to take up 1000 nm beads compared to control subjects. Defects in phagocytosis in patients with lung cancer have been reported previously, although these results have not specified differences in function between the different lung cancer cell types. Phagocytosis of opsonized sheep red blood cells was similar in AM obtained from patients with primary and metastatic lung malignancies [14] including patients with squamous, adenocarcinoma, large undifferentiated and oat cell carcinomas at varying clinical stages of disease. AM showed a decreased ability to phagocytoze bacillus Calmette-Guerin in lung cancer patients compared to tuberculin-positive patients even in the presence of recombinant IFN-γ [15]. We confirm differences in the phagocytic ability of AM from lung cancer patients, despite prior stimulation with LPS.

AM are able to secrete both pro-inflammatory and anti-inflammatory cytokines that influence the immediate microenvironment of T cells [16,17]. Pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 promote the induction of Th1 cells, which are essential for anti-tumour immunity. IL-6 has been shown to regulate the ability of AM in lung cancer patients to be stimulated by IFN-γ and LPS and to subsequently stimulate anti-tumour immunity. Anti-inflammatory cytokines such as IL-10 and TGF-β induce Th2/Tr1 cells which suppress anti-tumour immunity and promote tumour progression [9,18]. Tumor-associated macrophages from patients with non small cell lung carcinoma have been demonstrated to have reduced production of cytokines, IL-1, IL-6, TNF-α, TGF-β as well as reduced chemotactic activity, antibody-dependent cell-mediated cytotoxicity and cytostatic activity, similar to results reported in this study [17]. These same AM were, however, able to be induced in vitro to secrete increased amounts of these cytokines.

In the current study AM from patients with small cell carcinoma produced reduced amounts of all cytokines assayed, in keeping with previous results from our group and others [19]. IL-1 was also decreased overall in patients with non small cell lung cancer, although when subtypes were examined, there was no significant reduction in IL-1 secretion from the patients with adenocarcinoma, compared with controls. TNF-α secretion was reduced in AM from all lung cancer patients but IL-6 secretion was reduced only in the large cell subgroup of non small cell lung cancer. IL-10 is a cytokine with anti-inflammatory activity which has been shown to inhibit macrophage costimulation of T cells [20,21]. It is a potent inhibitor of tumour cytotoxic function of monocytes and AM but also a potent inhibitor of tumour angiogenesis [22]. Since IL-10 can alter the function and T cell activating activity of antigen presenting cells, we determined whether IL-10 was being produced by AM in this group of patients with lung cancer. Increased levels of IL-10 have been associated with a poor prognosis in patients with non small cell lung cancer [23–26]. However IL-10 was produced in comparable amounts by AM from cancer patients and control subjects in one study [27]. Previous studies have shown IL-10 production from unstimulated AM to be low or absent in control subjects, although production was increased to some extent after stimulation with LPS, and further still after costimulation with IFN-γ by AM and monocytes in sarcoidosis [28]. Previous authors have shown that IL-10 secretion by human AM (stimulated with LPS) peaks at 24 h [27]. We observed very low production of IL-10 by AM from control and adenocarcinoma subjects in our study, after stimulation of the AM for 18 h with LPS. There were no detectable levels of IL-10 (< 10 pg/ml) secreted from AM from patients with squamous, small and large cell undifferentiated carcinoma. It may be that he relatively low levels of IL-10 found in the current study are a reflection of the lower incubation time as we assayed supernatants after 18 rather than 24 h. At such low levels of IL-10 production it is difficult to draw firm conclusions about differential responses between cancer and noncancer groups. However, as with many of our other results, the adenocarcinoma subgroup differed minimally from the control subjects, while other non small cell lung cancer subgroups showed variable but significant differences from controls.

The clinical status of primary lung cancer patients may influence AM function. Previous reports have generally grouped primary lung cancer subtypes together or subdivided them only according to small cell or non small cell lung cancer subtypes, and have often described only differences between control and lung cancer groups. Patients who had received systemic therapy such as chemotherapy or radiotherapy, showed decreased levels of HLA-DR and Fc receptor [29]. However, patients on GM-CSF therapy showed an increased secretion of IL-1, IL-6 and TNF-α[30–32]. Although in some studies, IL-1, IL-6 and TNF-α are increased in lung cancer patients, these cytokines progressively decrease as the clinical stage of cancer progresses. Lung cancer is strongly associated with cigarette smoking and the potential for functional defects in AM in smokers is likely. IL-1, TNF-α and IL-6 which are important for cytostasis have been shown to be decreased in smokers compared to nonsmokers [6,33]. This could also be related to a decreased level of Fas protein detected in AM from lung cancer patients [34].

The phenotype of AM and their ability to be activated by LPS using multicolour flow cytometric analysis were investigated. The ability of AM to stimulate T cells is associated with molecules expressed on the surface of AM. An effective immune response against tumours requires an interaction of AM and T cells. Although T cell studies are limited, AM from lung cancer patients have been hypothesized to be down-regulated. Studies performed in mouse models have shown that mice injected with metastatic lung carcinoma suppressed ConA stimulated T cell responses after 5 days. AM from lung cancer patients have been shown to be cytotoxic against tumour cells [12]. Secondary signals, such as accessory function ICAM-1 of antigen presenting cells and interaction of costimulatory molecules CD40, CD83, CD86 and HLA-DR are important for T cell activation. Differences in up-regulation of costimulatory molecules CD80 and CD86 have been observed in AM from patients with allergies [35] however, this has not been assessed in primary lung cancer patients. Phenotypic analysis suggests that there is no difference between CD68 and HLA-DR expression in AM from lung cancer patients compared to controls however, a significant increase in CD11c, CD11b and CD11a was observed [11]. The ability of AM to stimulate T cells will include the phenotype of the cells. An effective immune response against tumours requires an interaction of AM and T cells.

We demonstrated that HLA-DR was significantly decreased on AM from small cell carcinoma patients but not on those from patients with large cell undifferentiated, squamous cell and adenocarcinomas. The adhesion marker CD54 (ICAM-1) was significantly decreased on AM from patients with large, squamous and small cell carcinoma but not on AM from adenocarcinoma patients compared to AM from control patients. The mannose receptor is highly expressed on macrophages and dendritic cells. This receptor binds to mannose residues expressed on the surface of bacteria and other organisms and has been shown to be important for their uptake [36]. We have used mannan to target antigens to the mannose receptor on macrophages and dendritic cells, for receptor-mediated uptake of antigens for processing and presentation to MHC class I and class II molecules [36–40]. The overall expression of mannose receptor on the surface of AM is not known. In this study, we observed relatively low mannose receptor expression levels on AM in the control group. The expression levels of mannose receptor on AM were significantly lower than for controls in squamous and small cell carcinoma lung cancer groups. The costimulatory marker, CD83, was significantly decreased in all groups except adenocarcinoma. Overall, AM from adenocarcinoma patients were not altered in cell surface expression of MHC class II, ICAM-1, mannose receptor and CD83 whereas AM from small cell carcinoma had decreased levels of all markers; squamous cell carcinoma had decreased levels of all markers except MHC class II and large cell carcinoma had decreased levels only for ICAM-1 and CD83.

It is clear from both the current studies and previous work that AM from patients with lung cancer are altered in function. The explanation for these alterations is unclear. Previous reports have generally grouped primary lung cancer subtypes together or subdivided them only according to small cell or non small cell lung cancer subtypes, and have often described only differences between control and lung cancer groups. In the current studies we have found differential abnormalities depending upon tumour cell type, with fewer abnormalities observed in the adenocarcinoma group and more frequent abnormalities in the small cell carcinoma group. Clinical status of primary lung cancer patients may also influence AM function, but we did not have access to tumour stage in this population of patients attending for bronchoscopy. Some previous studies have determined that alterations in functional status vary with extent of disease or disease stage, but others have not. Patients who had received systemic therapy such as chemotherapy or radiotherapy showed decreased levels of HLA-DR and Fc receptor [29]. However, patients receiving GM-CSF therapy showed an increase in IL-1, IL-6 and TNF-α [30]. Although in some studies IL-1, IL-6 and TNF-α are increased in lung cancer patients, these cytokines progressively decrease as the clinical cancer stage progresses. Lung cancer is strongly associated with cigarette smoking and the potential for functional defects in AM in smokers is likely. IL-1 [6], TNF-α and IL-6 which are important for cytostasis have been shown to be decreased in smokers compared to nonsmokers [33]. This could also be related to a decreased level of Fas protein detected in AM from lung cancer patients [34].

Several approaches to activate dysfunctional AM from murine lung cancer have been investigated. Stimulation of the CD40L on murine AM increased the production of nitric oxide, IL-12, TNF-α and anti-tumour immunity [41]. Antibodies against IL-6 and a synthetic peptide of C-reactive protein have been added to in vitro cultures of AM to induce increased IL-1α, IL-1β and TNF-α secretion after stimulation with LPS and IFN-γ and enhance tumoricidal activity against allogeneic lymphocytes [42–44]. These studies demonstrate the potential to re-activate dysfunctional AM in lung cancer patients.

Our findings show a clear defect in the ability to take up 40 nm and 1000 nm latex beads, decreased surface expression of HLA-DR, ICAM-1, CD83 and mannose receptor and reduced production of cytokines TNF-α, IL-1 and IL-6 on AM from primary lung cancer patients. These results suggest AM ability to stimulate T cell responses and to subsequently generate an anti-tumour immune response may be significantly compromized in many patients with lung cancer. Interestingly, not all AM primary lung cancer subtypes have altered function, with AM from patients with adenocarcinoma in this study presenting a profile quite similar to control subjects. Future studies will try to tease out tumour stage, smoking status, age and other variables which may be affecting AM functions differentially. Exploring the mechanisms underlying such defects further may enable us to develop strategies to enhance AM anti-tumour function and T cell immunity and thus improve prognosis in this disease.

Acknowledgments

This work was supported by the Carole Gabay Fund of the Institute of Breathing and Sleep. VA is an NH & MRC R. Douglas Wright Fellow 223316 and Sir Zelman Cowen Cancer Research Fellow. The authors would like to thank Prof Geoffrey Pietersz for helpful discussions.

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