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Abstract
Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have demonstrated impaired bacterial phagocytosis and disordered cytokine secretion, which are calcium-dependent processes. We determined how calcium moderates the macrophage response to nontypeable Haemophilus influenzae (NTHI). We hypothesized that augmenting extracellular calcium during bacterial challenge would restore bacterial phagocytosis and cytokine secretion in monocyte-derived macrophages (MDMs) from subjects with COPD. We further determined whether restoration of pattern recognition and scavenger receptors correlated with the calcium-induced improvements in macrophage function. Monocytes were purified from whole blood from healthy control subjects (n = 20) and patients with moderate to severe COPD (n = 35), and cultured in suspension with granulocyte macrophage colony–stimulating factor to generate MDMs. The MDMs were incubated with fluorescently labeled NTHI with and without calcium lactate and calcium channel inhibitors. Phagocytosis efficiency was evaluated by flow cytometry. Supernatants were assayed for cytokines using bead array technology. Cell surface receptor expression was assayed by multicolor flow cytometry. Extracellular calcium significantly improved phagocytosis and cytokine secretion (IL-8, TNF-α, and macrophage inflammatory protein [MIP]-1α, and -1β) in COPD MDMs. NTHI challenge led to statistically significant reductions in CD16 (FcγRIII), and extracellular calcium up-regulated both CD16 and macrophage receptor with collagenous structure (MARCO). Specific calcium channel inhibitors abrogated calcium-mediated MARCO up-regulation and cytokine secretion. Extracellular calcium improved phagocytosis, restored innate cytokine secretion, and increased cell surface expression of bacterial recognition receptors, CD16 and MARCO. These observations support the therapeutic use of calcium to improve macrophage function in COPD to decrease exacerbations and chronic bacterial infection.
Chronic obstructive pulmonary disease (COPD) is associated with frequent bacterial exacerbations and chronic bacterial infection. Impaired macrophage cytokine response and phagocytosis in alveolar and suspension-cultured monocyte–derived macrophages (MDMs) has been demonstrated in COPD and likely contributes substantially to the increased susceptibility for bacterial infection in these patients. We are the first to demonstrate that increases in calcium in the extracellular environment can restore demonstrated deficits in the innate immune response to bacterial pathogens in suspension-cultured MDMs from patients with COPD. If these findings are confirmed in bronchoscopically obtained alveolar macrophages, this would provide evidence for a therapeutic option that can alter the susceptibility to infection in COPD.
Despite augmented numbers of both neutrophils and macrophages in the airways of patients with chronic obstructive pulmonary disease (COPD), there is clear evidence that these patients are less able to clear infectious pathogens (1–6). Inability to clear bacterial pathogens is manifest in all stages of COPD as exacerbations, increased susceptibility to pneumonia, and chronic bacterial colonization of the airways. This chronic colonization leads to the development of a persistent neutrophilic presence in the airways (7–9), and this, coupled with recurrent exacerbation, has been associated with disease progression (9).
The alveolar macrophage functions as the first line of defense in the innate immune response to potential pathogens, and has been shown to have impaired functionality in patients with COPD, both in terms of impaired phagocytosis and an impaired ability to secrete cytokines responsible for activating the innate and adaptive immune responses (1–4, 6). It has been shown that alveolar macrophages have impaired bacterial phagocytosis (1–4, 6), and that this phagocytic process requires F-actin internalization and cytoskeleton rearrangement, is dependent on lipid rafts in the plasma membrane and receptor clustering to enhance substrate binding efficiency, and requires effective phagolysosome formation (4, 10, 11). These initial steps of bacterial recognition, internalization, and killing have been demonstrated to have a calcium requirement, and suggest a significant role for calcium in regulating the initial immune response to bacterial challenge.
Subsequent work on calcium-specific and nonspecific calcium-permeable cation channels (transient receptor potential vanilloid [TRPV] 2, transient receptor potential melastatin receptor [TRPM] 6, and transient receptor potential receptor, subfamily C [TRPC]), and their effects on intracellular calcium, has added to the growing body of data that calcium is integral in bacterial phagocytosis (11–15), and help identify the channels involved in the calcium oscillations required for an effective immune response. In addition to phagocytosis and phagolysosome function, calcium has also proven integral to secretion of cytokines associated with innate and adaptive immune responses (TNF-α, IL-2, IL-6, IL-8, IL-12, and IL-23) (16–22).
That the innate immune processes of bacterial phagocytosis and cytokine secretion are both impaired in COPD and are calcium dependent, provides the basis for the hypothesis and pilot study, that the modulation of extracellular calcium may provide one avenue to restore these aspects of the innate immune response. Using a suspension culture, granulocyte/macrophage colony–stimulating factor (GM-CSF)–stimulated monocyte-derived macrophage (MDM) model that more accurately represents the alveolar macrophage than adherence maturation models (6, 23), and is more readily accessible for preliminary experiments, we hypothesized that augmenting extracellular calcium in COPD at the time of bacterial challenge would restore both bacterial phagocytosis and cytokine secretion in MDMs from patients with COPD. We further sought to determine the mechanism by which the calcium-induced improvements were mediated.
All study procedures received institutional review board approval from the Department of Veterans Affairs Western New York Healthcare System. Informed consent was obtained from each participant before study entry. The study subjects were screened for inclusion into one of two groups: subjects with moderate to severe COPD and healthy control subjects. Additional inclusion and exclusion criteria are listed in the online supplement.
Monocytes were purified from whole blood by density centrifugation and negative bead depletion. Details are provided in the online supplement.
Monocytes were obtained as described previously here, and matured in suspension culture as described previously elsewhere (23). Details are provided in the online supplement.
Nontypeable Haemophilus influenzae (NTHI; 11P6H) is a sputum isolate obtained from a patient with COPD at exacerbation. Bacteria were fluorescently labeled with FITC. Details are provided in the online supplement.
Calcium lactate, with or without inhibitor treatment, was added to the MDM–bacterial cultures with a final projected multiplicity of infection (MOI) of 200:1 bacteria: cells and incubated at 37°C on a rotational shaker for 4 hours. Phagocytosis efficiency, cell surface receptor expression cytokine analysis, and intracellular calcium levels were evaluated at the end of the 4-hour coculture incubation period. Additional details are provided in the online supplement.
Phagocytosis efficiency was determined by FACS analysis (FACS Canto II; BD Biosciences, San Jose, CA) for median fluorescence intensity (MFI) in the FITC channel, and by percent of cells in the R1 gate demonstrating FITC fluorescence. Data analysis was done with FlowJo software (Treestar, Ashland, OR).
After a 4-hour incubation, aliquots of the bacteria–cell mixture were plated on chocolate agar. Bacterial colony counts were performed after 24 hours to determine effects of calcium on bacterial killing.
Intracellular calcium levels were assessed using a ratiometric analysis of Indo-1 fluorescence on a microplate fluorimeter (ThermoFisher Scientific, Waltham, MA). Extracellular calcium effects on intracellular calcium levels were assessed in unstimulated cells (see Figure E1 in the online supplement). Details are provided in the online supplement.
Supernatants from the bacteria–MDM coculture experiments were assayed by bead-based immunoassays using the CBA flex sets (BD Biosciences) for IL-1β, IL-6, IL-8, IFN-γ, IL-10, TNF-α, IL-12p40, macrophage inflammatory protein (MIP)-1α, MIP-1β, and monocyte chemoattractant protein (MCP)-1. Results were analyzed using FCAP Array software (BD Biosciences).
Multicolor flow cytometry for the cell surface receptors, CD14, CD93, CD35, Toll-like receptor (TLR) 4, CD16 (FcγRIII), TLR2, CD206, CD11b, CD1d, scavenger receptor A (SRA-1), and intracellular receptor TLR9, was performed on the MDMs. Details are provided in the online supplement.
MDM–bacteria incubations were performed as described previously here. Thirty minutes prior to NTHI challenge, calcium and select calcium channel inhibitors (diltiazem, ruthenium red, SKF93,635, spermine) were added to the media. Details are provided in the online supplement.
Statistical analysis was completed using Prism 6 (GraphPad Software Inc., San Diego, CA) and JMP software (Cary, NC), using nonparametric analyses. Details are provided in the online supplement.
Due to the limitation in the number of monocytes that could be obtained from each subject, the presence of an effect of calcium on the innate immune response in COPD was determined in the first 22 subjects (14 subjects with COPD, 8 healthy control subjects), whereas mechanistic studies were performed in the next 33 subjects (21 subjects with COPD, 12 healthy control subjects). Previous studies within our group on alveolar macrophages have demonstrated the ability to achieve statistically significant differences with similar group sizes (2, 3).
Demographic data for subjects were not statistically different between proof-of-concept and mechanistic arms of the study, and are presented in compilation form in Table 1. There were no statistically significant differences between ex-smokers and current smokers within the COPD group for degree of airflow limitation or cumulative tobacco exposure. Besides the expected differences in lung function, healthy control subjects were younger than the subjects with COPD (P < 0.0001). There were also more women in the healthy control group (P = 0.005).
| COPD Smokers | COPD Ex-Smokers | COPD All | Healthy Control Subjects | P Value | |
|---|---|---|---|---|---|
| No. of subjects | 15 | 20 | 35 | 20 | |
| Male/female, n | 12/3 | 19/1 | 31/4 | 10/10 | P = 0.005, COPD:HC |
| Age, mean (95% CI) | 63 (56–68) | 67 (65–81) | 66 (62–68) | 47 (37–54) | P < 0.0001, COPD:HC |
| P = 0.06, NS:S | |||||
| FEV1 %, mean (95% CI) | 52 (34–63) | 53 (43–66) | 51 (44–57) | 99 (91–107) | P < 0.0001, COPD:HC |
| P = 0.39, NS:S | |||||
| FEV1, L, mean (95% CI) | 1.5 (1.2–1.9) | 1.7 (1.4–2.0) | 1.6 (1.4–1.9) | 3.3 (3.1–3.7) | P < 0.0001, COPD:HC |
| P = 0.40, NS:S | |||||
| FEV1/FVC ratio (95% CI) | 47 (40–54) | 51 (45–57) | 49 (45–54) | 84 (77–92) | P < 0.0001, COPD:HC |
| P = 0.59, NS:S | |||||
| Cumulative tobacco exposure, pack-years (95% CI) | 53 (45–76) | 53 (41–72) | 53 (48–69) | 0 | P < 0.0001, COPD:HC |
| P = 0.58, NS:S |
Suspension-cultured MDMs from subjects with COPD demonstrated phagocytic deficits, when compared with healthy control subjects (Figure 1A). The suspension culture monocyte maturation model (6, 23), matured with GM-CSF, more accurately represented the innate immune deficits observed in bronchoscopically obtained human alveolar macrophages as compared with the published adherence-based maturation models (without GM-CSF), which did not replicate the phagocytic or cytokine deficits (2). These data support the use of the GM-CSF–matured, suspension culture MDM in vitro model as the best available, noninvasive model for mechanistic studies involving the alveolar macrophage.
Figure 1. Extracellular calcium improves bacterial phagocytosis of nontypeable Haemophilus influenza (NTHI) in monocyte-derived macrophages (MDMs) from subjects with chronic obstructive pulmonary disease (COPD) and restores phagocytosis to normal levels. (A) MDM from subjects with COPD demonstrated reduced phagocytic ability (flow cytometric analysis of median fluorescence intensity [MFI]) representing average intracellular uptake of FITC-labeled NTHI; #P = 0.036, top panel). Differences in phagocytosis as percentage of cells participating in phagocytosis (bottom panel) did not reach statistical significance (P = 0.09, not significant [NS]), suggesting differences may be related to more efficient per-cell bacterial uptake by healthy control subjects, with a trend toward increasing numbers of healthy control MDMs participating in phagocytosis. (B) The presence of 1 mM extracellular calcium improved phagocytosis of NTHI in MDMs from subjects with COPD, as assessed by MFI (top panel) and by percentage of cells participating in phagocytosis (bottom panel) (#P < 0.001 for both top and bottom panels). Calcium did not increase phagocytic efficiency of NTHI in healthy control subjects (P = 0.22, MFI; P = 0.30, percentage of phagocytosis). (C) Extracellular calcium restored phagocytosis of COPD-derived MDMs to normal levels, as assessed by MFI (top panel; P = 0.57, NS) and percentage of cells participating in phagocytosis (bottom panel; P = 0.86, NS). For all figures, the longer horizontal line represents the median value; error bars denote the interquartile range (IQR; n = 35, COPD; n = 15, healthy control).
[More] [Minimize]MDMs were incubated with live NTHI (11P6H) in the presence of Hanks’ balanced salt solution without calcium or magnesium, with the addition of calcium lactate, at concentrations within the range of 1–5 mM. These concentrations were chosen to encompass a range around the published physiologic calcium concentration of airway lining fluid (24, 25) and the ceiling at which crystallization of the calcium occurred or calcium-induced cellular toxicity developed in our ex vivo model system. Extracellular concentrations above 1 mM demonstrated concentration-dependent crystallization and cellular toxicity (P = 0.006, Freidman multicomparison analysis for decreased cell survival with increasing concentrations of extracellular calcium from 1 to 5 mM) in our system, which has also been observed recently in other macrophage cell lines at 5 mM (26).
The use of a control group of calcium-free media alone was chosen to determine the contribution of intracellular calcium store release in the response to NTHI, and is compared with the experimental conditions of additional extracellular calcium and extracellular calcium influx.
Phagocytosis of NTHI by MDMs from subjects with COPD improved when calcium lactate was added to the extracellular environment at a concentration of 1 mM (P < 0.001; Figure 1B). This statistically significant improvement was present when phagocytic efficiency was determined by MFI or as a percent of MDM participating in phagocytosis (% phagocytosis). There was a concentration-dependent improvement in phagocytosis to increasing calcium concentrations to 5 mM (P <0.0001, MFI; P = 0.005, % phagocytosis), but with a reduction in cell survival. Therefore, the 1 mM calcium concentration was selected for further investigation. Healthy control MDMs did not demonstrate statistically significant improvements in phagocytosis of NTHI in the presence of extracellular calcium (P = 0.21). In the presence of extracellular calcium, MDMs from subjects with COPD demonstrated phagocytic responses to NTHI that were not statistically different from the responses of untreated healthy control MDMs (P = 0.62; Figure 1C). Extracellular calcium did not alter the intracellular killing of phagocytosed bacteria (data not shown) for either subjects with COPD or healthy control subjects. This demonstrates that the presence of extracellular calcium at the time of NTHI challenge is sufficient to restore the phagocytic response in COPD-derived MDMs to normal levels.
Improvement in NTHI phagocytosis by MDMs from subjects with COPD in the presence of extracellular calcium was not diminished in the presence of chemical inhibitors of select calcium and cation channels (diltiazem, ruthenium red, spermine, and SKF96,365).
MDMs from subjects with COPD challenged with NTHI in the presence of extracellular calcium demonstrated significantly improved secretion of IL-8, TNF-α, MIP1α, and MIP1β (Figure 2). The presence of extracellular calcium restored cytokine secretion from COPD monocyte–derived macrophages to levels not statistically different from untreated healthy control responses to NTHI (Table 2). IFN-γ levels and IL-10 were below the limits of detection at baseline and after NTHI challenge, and did not increase in the presence of extracellular calcium. IL-1β, IL-6, and MCP-1 demonstrated improvements, but did not reach statistical significance. Extracellular calcium did not significantly alter cytokine secretion from healthy control MDMs.
Figure 2. Extracellular calcium improves cytokine secretion in MDMs from subjects with COPD. The presence of 1 mM extracellular calcium improved COPD-derived MDM cytokine secretion after NTHI challenge for IL-8 (P = 0.004), TNF-α (P < 0.0001), macrophage inflammatory protein (MIP)-1α (P = 0.0014), and MIP-1β (P = 0.004) (n = 31, COPD). Graphs in the right column demonstrate magnification of the lower concentrations. Calcium did not increase cytokine secretion from MDMs from healthy control subjects (n = 20). Cytokine values are reported (pcg/ml) and were analyzed after 4-hour NTHI exposure, in parallel to the phagocytosis endpoint.
[More] [Minimize]| COPD MDMs Calcium Treated | Healthy Control MDMs | ||
|---|---|---|---|
| Cytokine | pcg/ml (IQR) | pcg/ml (IQR) | P Value |
| IL-1β | 3 (0–10) | 3 (0–13) | 0.91 |
| IL-6 | 17 (0–77) | 12 (4–5,120) | 0.51 |
| IL-8 | 374 (206–1,846) | 919 (137) | 0.61 |
| TNF-α | 7 (0.3–1,884) | 850 (13–1,838) | 0.23 |
| MIP-1α | 21 (42–1,753) | 615 (54–1,230) | 0.19 |
| MIP-1β | 37 (2–1,103) | 448 (4–1,779) | 0.15 |
| MCP-1 | 9 (0–134) | 57 (0–105) | 0.56 |
In MDMs obtained from subjects with COPD, calcium-induced increases in secretion of IL-8 and MIP-1β were abrogated in the presence of ruthenium red (Table 3). Diltiazem also inhibited cytokine secretion, though limited to inhibition of TNF-α (Table 3). Chemical inhibition of calcium channels with subsequent reduction of cytokine response further supports the role for extracellular calcium in restoring cytokine elaboration as part of the innate immune response.
| COPD MDMs, Calcium Treated | COPD MDM, Calcium Treated, Ruthenium Red | COPD MDMs Calcium Treated Diltiazem | ||
|---|---|---|---|---|
| Cytokine | Median pcg/ml (IQR) | Median pcg/ml (IQR) | Median pcg/ml (IQR) | P Value |
| IL-8 | 286 (166–387) | 197 (103–309) | — | P = 0.0009 |
| MIP-1β | 9 (0–29) | 2.7 (0.005–24) | — | P = 0.04 |
| TNF-α | 0.9 (0.1–4.5) | — | 0 (0–0.15) | P = 0.001 |
The presence and relative density of receptors was determined by multicolor flow cytometric analysis. Of the various receptors studied, exposure to NTHI led to a significant reduction in CD16 (FcγRIII) by MFI (P = 0.048) on the surface of MDMs from subjects with COPD and healthy control subjects (P = 0.03) (Table 4). The percent of cells expressing CD16 also declined in MDMs from both healthy control subjects (P = 0.01) and subjects with COPD (P = 0.01) after NTHI exposure (Table 4).
| CD16-Untreated Cells | CD16 NTHI-Challenged | % Cells Expressing CD16 Untreated Cells | % Cells Expressing CD16 NTHI Challenged | |||
|---|---|---|---|---|---|---|
| Group | MFI (IQR) | MFI (IQR) | P Value | (IQR) | (IQR) | P Value |
| Subjects with COPD | 167 (100–214) | 121 (80–181) | 0.048 | 32 (20–46) | 30 (20.5–36.3) | 0.01 |
| Healthy control subjects | 6 (3–20) | 3.9 (3–9) | 0.03 | 27 (18–41) | 18 (12.7–33) | 0.01 |
The presence of 1 mM extracellular calcium at the time of NTHI exposure restored cell surface expression of CD16 in MDMs from subjects with COPD to baseline values (P = 0.03, MFI), but did not change the overall percent of COPD MDM expressing CD16 (P = 0.12). MDMs from healthy control subjects did not demonstrate statistically significant increases in CD16 expression in response to extracellular calcium (P = 0.08, MFI; P = 0.31, % of cells expressing CD16; Tables 5 and 6).
| MFI, NTHI Challenged | MFI, Calcium Treated, NTHI | |||
|---|---|---|---|---|
| Group | Receptor | (IQR) | (IQR) | P Value |
| Subjects with COPD | CD16 | 121 (80–181) | 135 (93–271) | 0.03 |
| MARCO | 280 (246–378) | 338 (231–434) | 0.01 | |
| Healthy control subjects | CD16 | 3.9 (3–9) | 7.2 (3–11) | 0.08 |
| MARCO | 50 (17–275) | 57 (19–387) | 0.005 |
| Receptor Expression NTHI Challenged | Receptor Expression Calcium Treated, NTHI | |||
|---|---|---|---|---|
| Group | Receptor | % (IQR) | % (IQR) | P Value |
| Subjects with COPD | CD16 | 30 (20.5–36.3) | 34.5 (20–40) | P = 0.12 |
| MARCO | 4.7 (2.9–7.3) | 5.4 (4.2–7.1) | P = 0.16 | |
| Healthy control subjects | CD16 | 18 (12.7–33) | 23 (13.3–33) | P = 0.31 |
| MARCO | 6.3 (3.9–9.4) | 4.4 (2.4–8.1) | P = 0.30 |
Extracellular calcium at the time of NTHI challenge increased macrophage receptor with collagenous structure (MARCO) expression by MFI in COPD MDM (P = 0.01) and healthy controls (P = 0.005) (Table 5). The magnitude of the median increase was substantially smaller in healthy control MDMs than that observed in COPD MDMs (MFI median increase of 118 in COPD and 7 in healthy control). Extracellular calcium did not change the overall percentage of COPD or healthy control MDMs expressing MARCO, supporting an increase in the per-cell receptor expression.
The only potentially negative effect of calcium was the reduction of cell surface expression of CD206 (macrophage mannose receptor; P = 0.04 [data not shown]) in MDMs from subjects with COPD. Extracellular calcium had no statistically significant effect on the other receptors studied (TLR2, TLR4, TLR9, CD35, CD93, CD11b, CD14, and CD1d) in MDMs from subjects with COPD or healthy control subjects.
Specific calcium and cation channel inhibitors (diltiazem, ruthenium red, spermine, and SKF93,635) did not alter the calcium-induced increases in CD16 expression in MDMs from subjects with COPD. The calcium-induced increase in MARCO expression on MDMs from subjects with COPD was inhibited in the presence of both diltiazem (P < 0.0001) and ruthenium red (P = 0.007; Table 7). MDMs from healthy control subjects also demonstrated a reduction in calcium-mediated increases in MARCO expression, although this was limited to only ruthenium red (P = 0.0005).
| MARCO Expression Calcium-Treated, NTHI | MARCO Expression Calcium-Treated, NTHI+RR | MARCO Expression Calcium-Treated, NTHI+Dilt | ||
|---|---|---|---|---|
| MFI (IQR) | MFI (IQR) | MFI (IQR) | P Value | |
| Subjects with COPD | 338 (231–434) | 220 (171–315) | 235 (196–312) | P < 0.0001, Dilt |
| P = 0.007, RR | ||||
| Healthy control subjects | 57 (19–387) | 49 (17–219) | 71 (16–211) | P = 0.0005, RR |
Incubation with NTHI led to reductions in relative intracellular calcium levels, as measured by ratiometric fluorescence of Indo-1 in MDMs of both subjects with COPD and control subjects (P = 0.007). The presence of 1 mM extracellular calcium led to increases in intracellular calcium (P = 0.038) to levels above baseline, normalizing the reduction induced by NTHI, and correlated with the improvements in phagocytosis and cytokine release (Figure 3).
Figure 3. Extracellular calcium restored intracellular calcium levels in NTHI-challenged MDMs. Baseline ratiometric calcium is noted in the unstimulated lane (bound:unbound median ratio = 9.65; IQR = 8.5–10.5). Challenge with NTHI led to statistically significant reductions in intracellular calcium (*P = 0.007, bound:unbound median ratio = 9.5; IQR = 8.3–10.2). The addition of extracellular calcium increased intracellular calcium levels above unstimulated levels (**P = 0.038; median ratio = 9.99; IQR = 8.8–11.2); n = 14 (11 healthy control subjects, 3 subjects with COPD). Calcium concentration is expressed as ratio of Indo-1 bound to calcium (405 nm emission) and unbound dye (485 nm emission). Subjects were pooled, as this was a proof of concept, that endpoint effects may be mediated by differences in intracellular calcium levels. The longer horizontal line represents the median value; error bars denote the IQR.
[More] [Minimize]There was no significant effect of age, FEV1, or smoking status on the improvements in phagocytosis, MARCO expression, or cytokine secretion, as assessed by multiple regression analysis, suggesting a more global disease-specific improvement.
Calcium has been shown to be integral as a second messenger in multiple signaling cascades (17, 18, 20, 22, 27–30). It has also been shown to be critical for effective phagocytosis of opsonin-dependent FcγR-mediated pathways (11, 14, 15), and in elaboration of inflammatory cytokines. Its role in opsonin-independent innate immune responses has not been as well studied; however, our data now suggest that calcium does participate in opsonin-independent responses to NTHI.
The effect of extracellular calcium on macrophage response to bacterial pathogens has been explored to a limited degree in the past. Previous literature suggested that calcium was not required for Fc receptor–mediated phagocytosis; however, more recent publications have demonstrated a significant role for both extracellular calcium entry and cell membrane– and phagolysosome-associated calcium channels in phagocytosis and phagosomal oxidative killing, as well as inflammatory cytokine secretion (11, 13, 15, 17, 22, 31–33). The more recent articles draw distinctions between the effects of calcium on the antigen-binding and internalization phases of phagocytosis.
Given the rapid oscillation of calcium signaling, small changes may be sufficient to restore immune effectiveness. The presence of extracellular calcium concentrations at near “physiologic” levels (24, 25) in our model was sufficient to restore the deficits in both phagocytosis and innate cytokine secretion. The use of a calcium-free media control group defined the extent of the cellular responses attributable to intracellular calcium stores (the untreated group). The calcium-free media control group from subjects with COPD demonstrated deficits in phagocytosis and cytokine secretion, relative to the calcium treated group, which suggests that the restorations of the innate immune responses of phagocytosis and cytokine secretion that were observed in the presence of extracellular calcium were mediated by influx of extracellular calcium. This supports our hypothesis and recent literature that extracellular calcium influx is responsible for the observed improvements in immune responses (11, 34).
The increases in cytokine secretion by MDMs from subjects with COPD in the presence of extracellular calcium increased only to levels consistent with the untreated healthy control response to NTHI challenge (Table 2). Increases in cytokine signaling did not appear to be indiscriminant and uncontrolled, with no effects on healthy control MDMs, supporting the hypothesis of a targeted correction of specific deficits related to COPD.
Mechanisms of the improvement in NTHI phagocytosis with calcium were investigated at the level of cell surface expression of pattern recognition receptors, including opsonin-independent TLR and scavenger receptors, as well as opsonophagocytic receptors for complement and IgG that have been identified as having some degree of plasticity in their target ligand. Extracellular calcium improved cell surface expression of CD16 and MARCO on MDMs from subjects with COPD. This increase in expression was due mostly to greater per-cell CD16 and MARCO expression (increase in MFI) without statistically significant increases in the percentage of cells expressing these receptors. This suggests that only a certain percentage of the MDMs from subjects with COPD is responsive to extracellular calcium in COPD. The up-regulation of CD16 (normally considered to be involved in opsonin-dependent pathogen recognition and phagocytosis) in our opsonin-independent model may reflect the recently identified ability of CD16 to directly bind carbohydrate moieties on gram-negative pathogens (35, 36), and thereby function in opsonin-independent phagocytosis. The association between up-regulation of MARCO, a scavenger receptor, and improved phagocytosis is more readily apparent, as MARCO has been identified as a major mediator of opsonin-independent phagocytosis in macrophages. MARCO has been shown to be present in reduced levels in alveolar macrophages from subjects with COPD, as well as macrophages from patients with cystic fibrosis, another disease in which impaired bacterial clearance has been established (37, 38). In a similar bacterial challenge model, treatment-induced improvements in cell surface MARCO levels (sulforaphane) were associated with improvements in bacterial phagocytosis in MDMs from subjects with COPD (38), further supporting the significance of MARCO early in the innate immune response. Future inhibitor studies will be required to determine if CD16 and MARCO are participating independently or synergistically in opsonin-independent macrophage phagocytosis.
The role of extracellular calcium influx in the improvements in MDM responses to NTHI is supported both by the data demonstrating sustained changes in intracellular calcium levels in the presence of extracellular calcium (Figures 3 and E1), as well as the inhibition of many of the improvements in the presence of select calcium channel inhibition. In the presence of ruthenium red, MDMs from subjects with COPD demonstrated inhibition of MARCO up-regulation, and inhibition of increases in IL-8 and MIP1β. Diltiazem also inhibited MARCO up-regulation, and selectively inhibited increases in TNF-α secretion. Increased CD16 expression and improvements in phagocytosis were not abrogated by either diltiazem or ruthenium red, suggesting that these two responses may be interrelated and distinct from the calcium-mediated improvements in cytokine secretion. There are limited data in neutrophils that influx of extracellular calcium is required for β2-integrin cross-linking (39) and that Fcγ receptors can be rapidly mobilized, within minutes of ligand binding (FcγRI/CD64) via β2-integrin cross-linking (40). Further study is required to determine if extracellular calcium influx rapidly mobilizes the CD16 receptor to the surface during the minutes before the inhibitors were added. The lack of inhibition of phagocytosis may be explained in part by the issues related to rapid CD16 up-regulation, as noted previously here, as well as redundancy to channels that participate in calcium influx and the resulting initial membrane depolarization required for initiating phagocytosis.
The inhibition of calcium-augmented IL-8, MIP1β, and TNF-α release by chemical blockade of calcium channels (ruthenium red inhibiting IL-8 and MIP1β and diltiazem inhibiting TNF-α) may be due to selective effects of the two inhibitors on different NF-kB signaling pathways (41, 42), or related to the degree of reduction of intracellular calcium levels (ruthenium red demonstrating greater reductions in intracellular calcium levels than diltiazem). Ruthenium red has also recently been shown to inhibit not only the TRP family of cation/calcium channels on the cell surface, but also the ryanodine receptors on the endoplasmic reticulum, which participate in calcium mobilization from the endoplasmic reticulum. This may explain the more potent lowering of intracellular calcium levels observed (Figure E2), as ruthenium red may inhibit both extracellular calcium influx and release of intracellular calcium stores. The calcium-mediated increase in release of IL-8 and TNF-α may be due to increased ADAM17/tumor necrosis converting enzyme activity induced by increasing intracellular calcium concentration. ADAM17 metalloprotease activity has been directly linked to TNF-α release from the precursor cell surface–bound form, and indirectly increases IL-8 release (43, 44). The 4-hour end point suggests calcium induced effects on mobilization of preformed cytokine stores, as there has been insufficient time for gene transcription and de novo protein translation.
Our data also suggest that the improvement in innate immune function of MDM may be related to restoration of intracellular calcium levels that are lowered by exposure to NTHI. Reduction in intracellular calcium is hypothesized to represent an attempt at immune evasion by reducing the magnitude of the immune response. There is a small, but growing, body of literature for immune evasion strategies of NTHI that lead to airway colonization and bacterial persistence, such as biofilm formation and prolonged intracellular survival (4, 8, 45–47). The ability to reduce intracellular calcium levels to evade immune detection or decrease phagolysosome formation and macrophage reactive oxygen species killing is used by other bacterial pathogens as well, including Mycobacterium tuberculosis and Listeria (48–50).
Though some our investigations were limited by cell recovery per patient, the restoration of key aspects of the innate immune response to bacterial infection in COPD-derived MDMs are provocative, and merit further investigation. These findings need to be replicated, and mechanistic studies expanded, in larger studies with bronchoscopically obtained alveolar macrophages. Suspension-cultured MDMs cannot entirely replicate the geographic effects of environment and cell–cell interactions of the alveolar macrophage. If our findings are substantiated in bronchoscopically obtained alveolar macrophages, this would provide evidence for a therapeutic option that may alter the susceptibility to infection in COPD.
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This work was supported by investigator-initiated grant funding from Pulmatrix, Inc.
This material is the result of work supported with resources and the use of facilities at the Department of Veterans Affairs Western New York Healthcare System (Buffalo, NY).
Author Contributions: K.A.P. was responsible for the design of the study, patient enrollment and recruitment, analysis and interpretation of data, manuscript preparation and submission; M.S. was responsible for optimization of experimental protocols, the acquisition of data, and assisted in the methods section of the manuscript preparation; S.P.A. assisted in the design of the study, data analysis and interpretation of data; D.L.H. assisted in the design of the study, data analysis and interpretation of data; S.S. assisted in the design of the study, analysis and interpretation of data, manuscript preparation and submission.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2014-0172OC on October 22, 2014
Author disclosures are available with the text of this article at www.atsjournals.org.
