help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on August 14, 2008, doi:10.1164/rccm.200804-646OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200804-646OCv1
178/9/894    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Calabrese, F.
Right arrow Articles by Saetta, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Calabrese, F.
Right arrow Articles by Saetta, M.
American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 894-901, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200804-646OC

Original Article

IL-32, a Novel Proinflammatory Cytokine in Chronic Obstructive Pulmonary Disease

Fiorella Calabrese1, Simonetta Baraldo2, Erica Bazzan2, Francesca Lunardi1, Federico Rea2, Piero Maestrelli3, Graziella Turato2, Kim Lokar-Oliani2, Alberto Papi4, Renzo Zuin2, Paolo Sfriso5, Elisabetta Balestro2, Charles A. Dinarello6 and Marina Saetta2

1 Department of Medical Diagnostic Sciences and Special Therapies, University of Padova; 2 Department of Cardiac, Thoracic, and Vascular Sciences, University of Padova and Padova City Hospital; and 3 Department of Environmental Medicine and Public Health, University of Padova, Padova, Italy; 4 Department of Clinical and Experimental Medicine, University of Ferrara, Ferrara, Italy; 5 Department of Clinical and Experimental Medicine, University of Padova, Padova, Italy; and 6 University of Colorado Health Sciences Center, Denver, Colorado

Correspondence and requests for reprints should be addressed to Marina Saetta, M.D., Università degli Studi di Padova, Dipartimento di Scienze Cardiologiche, Toraciche e Vascolari, Unità Operativa di Pneumologia, Via Giustiniani 3, 35128 Padova, Italy. E-mail: marina.saetta{at}unipd.it


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disorder of the lung, yet the mechanisms that regulate this immune-inflammatory response are not fully understood.

Objectives: We investigated whether IL-32, a newly discovered cytokine, was related to markers of inflammation and clinical progression in COPD.

Methods: Using immunohistochemistry, expression of IL-32 was examined in surgically resected specimens from 40 smokers with COPD (FEV1 = 39 ± 4% predicted), 11 smokers with normal lung function, and 9 nonsmoking control subjects. IL-32 was quantified in alveolar macrophages, alveolar walls, bronchioles, and arterioles, and confirmed by molecular analysis. The levels of IL-32 were correlated with the cellular infiltrates, markers of inflammation, and clinical data.

Measurements and Main Results: Macrophage staining for IL-32 was increased in smokers with COPD compared with control smokers and nonsmokers (P = 0.0014 and P < 0.0001, respectively), and similar differences were observed in alveolar walls (P = 0.0004 and P = 0.0005) and bronchiolar epithelium (P = 0.004 and P = 0.0009). This increase was also detected at the mRNA level (P = 0.007 vs. control smokers and P = 0.029 vs. nonsmokers) and was mainly due to non-{alpha} isoforms. Moreover, IL-32 expression was positively correlated with tumor necrosis factor-{alpha} (P = 0.004, rs=0.70), CD8+cells (P = 0.02, rs=0.46), phospho p38MAPK (P < 0.01, rs=0.60) and negatively with FEV1 values (P = 0.004, rs= –0.53).

Conclusions: This is the first study to demonstrate increased expression of IL-32 in lung tissue of patients with COPD, where it was colocalized with tumor necrosis factor-{alpha} and correlated with the degree of airflow obstruction. These results suggest that IL-32 is implicated in the characteristic immune response of COPD, with a possible impact on disease progression.

Key Words: inflammatory cytokines • immune response • airflow limitation • cigarette smoking



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Chronic obstructive pulmonary disease (COPD) is characterized by an exaggerated immune response, but the mechanisms of this response are yet unknown. IL-32 has recently been proposed as a possible regulator of innate and adaptive responses, particularly in inflammatory diseases.

What This Study Adds to the Field
IL-32 protein and mRNA were increased in lungs of smokers with COPD compared with unaffected subjects. IL-32 correlated with tumor necrosis factor-{alpha}, CD8+ cells, and phospho p38 mitogen-activated protein kinase, suggesting that this cytokine contributes to the characteristic immune response in COPD.

 
Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Estimates from the World Health Organization Global Burden of Disease indicate that, in 2001, COPD was the fifth leading cause of death in high-income countries and the sixth in nations of low and middle income, accounting for about 4% of total deaths (1). It is generally agreed that COPD is a progressive disease arising from an inflammatory response to noxious particles or gases (2). COPD is a classic gene-by-environment disease with various manifestations that reflect both the individual susceptibility and the degree of exposure to irritants, of which cigarette smoke is the most frequent. A number of studies have demonstrated that, in patients with COPD, a chronic inflammatory process is present throughout the airways, lung parenchyma, and pulmonary vasculature, and extends even outside the lung (3–6). The inflammatory response in the lung is characterized by infiltration of CD8+ T lymphocytes, which are polarized toward a type 1 profile with production of IFN-{gamma}, among other cytokines (7, 8).

IL-32 is a recently described cytokine produced by T lymphocytes, natural killer cells, monocytes, and epithelial cell lines (9, 10). IL-32 is prominently induced by IFN-{gamma} in vitro (9) and, conversely, its depletion reduces IFN-{gamma} production (11), thus suggesting a regulatory feedback mechanism. The gene encoding IL-32, which is organized into eight exons, is located on human chromosome 16p13.3; six splice variants have been described (IL-32{alpha}, IL-32β, IL-32{gamma}, IL-32{delta}, {varepsilon} and {zeta}) (9, 12), of which IL-32{gamma} is the full-length isoform without any exonic deletions. IL-32 exhibits several properties typical of proinflammatory cytokines. For example, the cytokine induces tumor necrosis factor (TNF)-{alpha}, IL-1β, IL-18, and chemokines through the activation of nuclear factor (NF)-{kappa}B and p38 mitogen-activated protein kinase (MAPK) (9).

IL-32 has recently been proposed as a possible regulator of innate and adaptive immune responses in vitro (10). In humans, only two in vivo studies have demonstrated up-regulation of IL-32, one in rheumatoid arthritis (13) and one in Crohn's disease (14), both of which have a pathogenetic autoimmune component. Whether this cytokine is implicated in the immune response characteristic of COPD still remains to be investigated. To explore this issue, in the present study the levels of IL-32 protein and mRNA were determined in surgically resected specimens from the following three groups of subjects: smokers with COPD, asymptomatic smokers with normal lung function, and nonsmoking control subjects. Morphometric analysis was also applied to quantify the amount of TNF-{alpha} in the same lung specimens, and colocalization of IL-32 and TNF-{alpha} was confirmed by confocal microscopy. Moreover, the expression of IL-32 was correlated with phosphorylated p38 MAPK, with the number of neutrophils and CD8+ cells infiltrating the alveolar walls and with the degree of airflow obstruction. Preliminary results of this study have been previously reported in abstract form (15, 16).


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subject Characteristics
To quantify the expression of IL-32 and TNF-{alpha}, we collected peripheral lung tissue from 60 subjects undergoing surgery for appropriate clinical indications: lung transplantation or lung volume reduction surgery for severe emphysema, and lung resection for solitary peripheral nodules (details are included in the online supplement). The subjects were categorized into the following three groups: smokers with COPD (GOLD [Global Initiative for Chronic Obstructive Lung Disease] stages I–IV; n = 40); asymptomatic smokers with normal lung function (control smokers; n = 11) and asymptomatic, nonsmoking subjects with normal lung function (nonsmokers; n = 9). Moreover, to look into disease specificity, IL-32 expression was evaluated in other inflammatory lung diseases of different etiologies. In particular, we examined autoptic samples from two subjects with asthma and two subjects with either viral or fungal pneumonia (cytomegalovirus or Aspergillus), as well as open lung biopsies from two subjects with collagen disease–associated nonspecific interstitial pneumonia (NSIP) pattern, and one subject with pulmonary involvement of rheumatoid arthritis.

Subjects with COPD did not experience any exacerbations or acute upper respiratory tract infections during the month preceding surgery. Each patient underwent interview, electrocardiography, routine blood tests, and pulmonary function tests, which were performed as previously described (17). The study conformed to the Declaration of Helsinki and was approved by the local ethics committee. Informed written consent was obtained for each subject undergoing surgery.

Immunohistochemistry and Morphometric Analysis
Lung tissue preparation and immunohistochemistry were performed as described in the online supplement. Briefly, sections were treated for 60 minutes with primary antibodies (at a concentration of 0.3 µg/ml) (i.e., murine anti-human IL-32 [clones 09 and 07, produced as previously described]) (11, 18) and anti-human TNF-{alpha} (T6817; Sigma-Aldrich, St. Louis, MO). IL-32 and TNF-{alpha} expression was quantified in alveolar macrophages and alveolar walls as described in the online supplement. To standardize the results, cell counts were expressed as percentage of positive macrophages over total macrophages examined, and as number of positive cells per millimeter of alveolar wall, respectively. Expression of IL-32 and TNF-{alpha} was also evaluated in peripheral airways (epithelium and smooth muscle) and pulmonary arterioles (tunica media) using a semiquantitative score (0: no staining; 1: weak staining; 2: moderate staining; 3: strong staining).

Confocal microscopy (details are included in the online supplement) was applied to confirm the coexpression of IL-32/TNF-{alpha} and IL-32/CD8 already observed in subsequent serial sections.

Moreover, to investigate potential correlations between IL-32 expression and other inflammatory parameters known to be up-regulated in COPD, we used immunohistochemical quantification of CD8+ T lymphocytes, neutrophils, and phospho p38+ cells obtained in previous reports (4, 19–22). Data for CD8+ T lymphocytes were available in 35 of the 60 patients, whereas data for neutrophils and phospho p38+ cells were available in 28 of 60 patients (equally represented among the different groups examined). Details are included in the online supplement.

Molecular Analysis for IL-32 mRNA Detection
Reverse transcriptase–polymerase chain reaction (RT-PCR) and semiquantitative evaluation of mRNA levels were performed in a representative population (nearly 50% of subjects included in the immunohistochemical analysis, equally distributed among the three groups examined): 17 patients with COPD, 7 control smokers, and 6 nonsmokers. All IL-32 PCR products were analyzed by gene sequencing. Further details are reported in the online supplement.

Statistical Analysis
All cases were coded and the measurements were made without knowledge of clinical data. Group data were expressed as mean and SEM, or as median and range when appropriate. Differences between groups were analyzed using the following tests for multiple comparisons: the analysis of variance and the unpaired Student's t test for clinical data and molecular findings, and the Kruskal-Wallis test and the Mann-Whitney U test for morphologic data. IL-32 isoform frequencies were compared by Fisher's exact test. Correlation coefficients were calculated using Spearman's rank method and corrected for multiple comparison using the Holm method (23, 24).


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical Characteristics of Study Subjects
The clinical characteristics of the subjects examined are shown in Table 1. Demographic analysis revealed that age was not significantly different in the three groups of subjects. Moreover, smoking history was similar in smokers with COPD and control smokers. As expected from the selection criteria, subjects with COPD had significantly lower values of FEV1 % predicted and FEV1/FVC % as compared with control smokers and nonsmokers. Among patients with COPD, 21 were in GOLD stage IV, 7 were in GOLD stage III, 10 were in GOLD stage II, and 2 in GOLD stage I. In smokers with COPD, the values of PaO2 were significantly reduced, and those of PaCO2 were significantly increased compared with the other two groups of subjects examined. Smokers with COPD had signs of lung hyperinflation (increased residual volume) and impaired carbon monoxide diffusion capacity (decreased DLCO) as compared with control smokers.


View this table:
[in this window]
[in a new window]

  		TABLE 1. PATIENT CHARACTERISTICS

 
Smokers with mild/moderate COPD, asymptomatic smokers with normal lung function, and asymptomatic, nonsmoking subjects with normal lung function did not receive antiinflammatory therapy (e.g., oral or inhaled corticosteroids) or antibiotics within the month preceding surgery, or bronchodilators within the previous 48 hours. All patients with very severe or severe COPD were treated with inhaled anticholinergics and/or β2-agonists/inhaled corticosteroids, and 10 of them with oral steroids.

Immunohistochemical Findings
IL-32 immunoreactivity was mainly observed in alveolar macrophages and alveolar walls (Figures 1A–1C). Positive staining was mostly detected at the cytoplasmic level with both diffuse as well as granular patterns. Strong nuclear staining was also observed, particularly in cuboidal alveolar cells (as characterized by anti-human cytokeratin, clone MNF116) of patients with severe COPD (Figures 1C and 1D). Extensive IL-32 immunoreactivity was also seen in peripheral airways (Figure 1B), particularly in the epithelium (as cytoplasmic staining in ciliated cells and in marginal areas of goblet cells) and in interstitial cells, including inflammatory cells, infiltrating the airway wall. The same IL-32 immunoreactivity was observed in central airways of patients with COPD, mainly represented in the epithelial layer and in bronchial glands (see Figure E2 in the online supplement).


Figure 1
View larger version (123K):
[in this window]
[in a new window]

  		Figure 1. (A–D) Immunohistochemistry for IL-32 in end-stage chronic obstructive pulmonary disease (COPD). (A) Strong cytoplasmic positivity was seen in all macrophages (arrows). (B) Besides macrophages (arrow), immunoreactivity was also detected in bronchiolar epithelial cells (arrowhead) and in interstitial inflammatory cells (arrowhead). (C) Strong nuclear (arrow) and cytoplasmic staining in alveolar walls (well evident in bottom insert). (D) Cytoplasmic positivity was also detected in cuboidal alveolar cells (arrows). Weakly positive alveolar macrophages (arrows) can be observed in the (E) smoking subject and (F) nonsmoking control subject. Original magnification = x200 (bottom insert: x400).

 
When quantitative analysis was performed, the percentage of IL-32+ macrophages was increased in smokers with COPD compared with smoking (P = 0.0014) and nonsmoking control subjects (P < 0.0001) (Figures 2A and 1A, 1E, and 1F). Patients with COPD were also grouped according to disease severity. Subjects with severe COPD were clustered with a markedly high percentage of IL-32+ cells, whereas patients with mild/moderate COPD exhibited scattered values; a trend was present for increased IL-32 expression in patients with severe COPD compared with those with mild/moderate disease (P = 0.07). Moreover, the percentage of IL-32+ macrophages was increased in smokers with normal lung function when compared with nonsmoking control subjects (P = 0.03) (Figure 2A).


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Figure 2. Individual counts for: (A) percentage of IL-32+ macrophages; (B) number of IL-32+ cells in alveolar walls; (C) percentage of tumor necrosis factor (TNF)-{alpha}+ macrophages; and (D) number of TNF-{alpha}+ cells in alveolar walls in smokers with chronic obstructive pulmonary disease (COPD), control smokers, and nonsmokers. Closed circles represent mild/moderate COPD, whereas open circles represent severe/very severe COPD. Horizontal bars represent median values. P values represent Mann-Whitney U test analyses. Overall comparison using Kruskal-Wallis test: P < 0.0001 for (A–C) and P = 0.05 for (D).

 
In alveolar walls, increased IL-32 expression was observed in smokers with COPD compared with both smoking and nonsmoking control subjects (P = 0.0004 and P = 0.0005, respectively) (Figure 2B). IL-32 expression was also increased in the bronchiolar epithelium of smokers with COPD when compared with control smokers (P = 0.004) and nonsmokers (P = 0.0009) (Table 2 and Figure 1B).


View this table:
[in this window]
[in a new window]

  		TABLE 2. SEMIQUANTITATIVE SCORES FOR IL-32 AND TUMOR NECROSIS FACTOR-{alpha}

 
No differences were found in IL-32 expression in smooth muscle of peripheral airways and pulmonary arterioles (Table 2). Moreover, in none of the compartments examined was IL-32 expression different between current or ex-smokers.

TNF-{alpha} immunostaining in alveolar macrophages was mainly observed in cytoplasm. The percentage of TNF-{alpha}+ macrophages was increased in both smokers with COPD and smokers with normal lung function when compared with nonsmokers (P < 0.0001 and P = 0.0005, respectively) (Figure 2C). Moreover, in alveolar walls, increased TNF-{alpha} expression was observed in smokers with COPD compared with nonsmoking control subjects (P = 0.018) (Figure 2D). No differences were seen in TNF-{alpha} expression between smokers with severe COPD and those with mild/moderate disease.

No differences were found in TNF-{alpha} expression in peripheral airway epithelium and smooth muscle, and in pulmonary arterioles (Table 2). In none of the compartments examined was TNF-{alpha} expression different between current or ex-smokers.

Confocal microscopy analysis showed coexpression of IL-32 and TNF-{alpha} in many macrophage-like cells (Figures 3A–3C) and metaplastic epithelial cells. CD8+ T-lymphocytes, which are the predominant cells infiltrating the alveolar walls in COPD, also coexpressed IL-32 (Figures 3D–3F).


Figure 3
View larger version (52K):
[in this window]
[in a new window]

  		Figure 3. Immunofluorescence laser scanning microscopy analysis showed the coexpression of IL-32 (A; red) and TNF-{alpha} (B; green) in the same macrophages. (C) The overlay image of (A) and (B) (arrows). Immunofluorescence laser scanning microscopy analysis also showed the presence of IL-32 (D; red) on CD8+ T cells (E; green). (F) The overlay image of (D) and (E) (arrows). Scale bars = 10 µm.

 
IL-32 evaluation in other inflammatory disorders of the lung revealed strong reactivity in the patient with rheumatoid arthritis and in those with collagen disease–associated NSIP, particularly in alveolar macrophages and, to a lesser extent, in alveolar walls (Figures 4A and 4B). By contrast, rare immunoreactivity for IL-32 was present in subjects with asthma and in those with pulmonary infections, whose samples were nearly negative (Figures 4C and 4D).


Figure 4
View larger version (158K):
[in this window]
[in a new window]

  		Figure 4. IL-32 evaluation in other inflammatory lung diseases: strong immunostaining was observed in (A) rheumatoid arthritis and (B) collagen disease–associated nonspecific interstitial pneumonia fibrosis pattern. Weak immunoreactivity, limited to alveolar macrophages, was present in (C) asthma and in (D) viral pneumonia. Arrows indicate IL-32–positive macrophages. Original magnification = x50.

 
Because most of the patients included in this study (either in COPD or in control groups) had lung cancer, there is concern that the concomitant presence of lung cancer may have influenced the results. However, among patients with lung cancer, those with COPD had an increased expression of IL-32 compared with those without COPD (P = 0.02 for IL-32+ macrophages and P = 0.007 for IL-32+ cells/mm). In addition, among patients with COPD, those with severe disease (the majority of which did not have lung cancer) showed a trend for increased IL-32 expression when compared with those with mild/moderate COPD (who had concomitant lung cancer).

Molecular Findings
The intensity of the band coding for glyceraldehyde-3-phosphate dehydrogenase in each sample did not differ significantly within the study groups. A significant increase of IL-32 mRNA was observed in smokers with COPD compared with both smoking and nonsmoking control subjects (4-fold [P = 0.007] and 2.8-fold [P = 0.03], respectively). No significant differences were detected between smoking and nonsmoking control subjects (Figure 5).


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		Figure 5. (A) MetaPhor gel electrophoresis for reverse transcriptase–polymerase chain reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and IL-32 non-{alpha} (β, {gamma}, and {delta}) mRNAs in emblematic cases. GAPDH is detected as a 130-bp amplicon (lanes 2, 4, 6, 8); IL-32{alpha} as a 136-bp amplicon (lane 3); and IL-32 non-{alpha} as a 307-bp amplicon (lanes 3, 5, 7, 9). Lane 1: DNA molecular weight marker. (B) Quantification of IL-32 non-{alpha} (β, {gamma}, and {delta}) mRNA expression in the three groups of patients (17 with COPD, 7 control smokers, and 6 nonsmokers). Shown data are the mean ± SEM; P values represent t test analysis.

 
The greater proportion of IL-32 mRNA was detected as a 307 base pair amplicon, corresponding to IL-32 non-{alpha} (β, {gamma}, and {delta}). A well-visible 136-bp PCR product, corresponding to IL-32{alpha}, was seen in 54% of control cases (7/13) and in only 6% of patients with COPD (1/17) (P = 0.009). Sequence analysis of all the amplicons showed a high homology (100%) with the expected IL-32 non-{alpha} (β, {gamma}, and {delta}) isoforms (accession numbers: NM004221, NM001012718, NM001012631, NM001012632, NM001012634, NM001012635, NM001012636) and IL-32{alpha} (accession number: NM001012633).

Correlations
When all patients were considered together, several statistically significant correlations were observed relating the different morphometric measurements to each other, and also to functional parameters. All details of this analysis are reported in the online supplement, including figures (Figures E3A and E3B). In particular, IL-32 expression was negatively correlated with lung function parameters (FEV1 and FEV1/FVC) and positively correlated with TNF-{alpha} expression, with the number of CD8+ cells infiltrating the alveolar walls and with the phosphorylation state of p38 MAPK. The correlations between IL-32 expression and FEV1, FEV1/FVC, TNF-{alpha}, and p38 MAPK remained significant when nonsmoking subjects were excluded from the analysis.


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first study that demonstrates increased IL-32 protein levels in lung samples from smokers with COPD compared with unaffected subjects. Elevated steady-state mRNA was consistent with the increase in IL-32 by immunohistochemistry. Moreover, IL-32 protein significantly correlated with the presence of TNF-{alpha}, with the number of CD8+ cells, and with the levels of phospho p38 MAPK.

It is now widely accepted that activation of an immune-inflammatory response plays a key role in the pathogenesis of COPD. An inflammatory response is present in virtually all smokers (25, 26), and this is thought to represent a normal, nonspecific response to injury (innate response) (27). However, not all smokers develop COPD, suggesting that the disease requires both exposure to cigarette smoke and individual susceptibility. It has been hypothesized that susceptibility to COPD may occur from a shift in the nonspecific innate response toward an adaptive immune response (28). Although the source of the specific antigen is still a matter of ongoing debate, it has been proposed that autoimmune mechanisms could be operational in COPD (29). Indeed, some recent studies have reported the presence of autoimmunity in smoking-induced emphysema by conventional criteria, demonstrating both cellular and humoral responses against self-antigens, particularly elastin peptides (30, 31).

To date, overexpression of IL-32 has been described in inflammatory disorders associated with an autoimmune component, but information on activation of this pathway in vivo is indeed limited. Up-regulation of IL-32 is present in synovial tissue of patients with rheumatoid arthritis (13), where it is correlated with the expression of proinflammatory cytokines, such as IL-1β, IL-18, and TNF-{alpha}, and with markers of clinical severity. Moreover, epithelial expression of IL-32 is enhanced in the inflamed mucosa of patients with Crohn's disease (14). Of interest, in our study, we observed intense IL-32 immunoreactivity, aside from COPD, in the patient with rheumatoid arthritis and in those with collagen diseases–associated NSIP, but not in the subjects with asthma or infective pneumonia. These observations suggest that IL-32, rather than a general signal, which becomes activated in each inflammatory state, could be more specifically associated with autoimmune responses.

In this study, we observed a prominent cytoplasmic staining in alveolar macrophages where IL-32 induces the production of several cytokines thought to be important in COPD, such as IL-1β, TNF-{alpha}, IL-6, and IL-18 (9). Of interest, down-regulation of these inflammatory mediators has been described using specific small inhibitory RNA against IL-32 in human peripheral blood mononuclear cells (11). In addition, small inhibitory RNA inhibiting IL-32 also suppresses nuclear binding of NF-{kappa}B and activator protein-1 (11). Notably, IL-32 expression was positively correlated with both the phosphorylation of p38 MAPK and TNF-{alpha} levels. These correlations, together with reports that NF-{kappa}B is activated in COPD (32), support the hypothesis that the IL-32/TNF-{alpha} pathway plays a key role in the amplification of the immune response.

The major known sources of IL-32 are inflammatory cells, particularly macrophages, which have been shown to produce this cytokine in vitro (10, 33). We demonstrated, for the first time in human lung tissue, an active IL-32 transcription confirmed by gene sequencing. The non-{alpha} IL-32 isoforms were abundant in patients with COPD. By contrast, the {alpha} isoform, which was prevalent in unaffected subjects, was hardly detectable in patients with COPD (only 1 out of 17 subjects). The functions of the different IL-32 isoforms are unknown; however, as with other cytokines, it is possible that the different splice variants may function as antagonists (34, 35). In line with these observations, in the present study, overexpression of the non-{alpha} isoforms in COPD is associated with depletion of the {alpha} isoform, even if it remains to be established whether the lack of the {alpha} isoform is detrimental.

In addition to alveolar macrophages, a prominent immunostaining was observed in the alveolar walls, particularly in cuboidal alveolar cells of patients with severe COPD. These cells exhibited marked cytoplasmic staining, in some cases associated with nuclear positivity. The significance of nuclear positivity in this cytokine, believed to be largely produced at the cytoplasmic level, is unknown. However, it is possible that nuclear translocation could influence the transcription of other cytokines and growth factors, as could be the specific case for TNF-{alpha}.

Moreover, other possible sources of IL-32 in the alveolar walls could be represented by CD8+ T lymphocytes that have the potential to produce IL-32 in vitro (12). In our study, we were able to demonstrate colocalization of IL-32 and CD8 by confocal microscopy, an observation that is further supported by the positive correlation observed between IL-32 expression and CD8+ cells in alveolar walls. Of interest, CD8+ T lymphocytes, which infiltrate the alveolar walls in patients with COPD (4, 21, 36), may promote apoptosis of epithelial cells through different mechanisms that may also require IL-32 (14). Indeed, extensive alveolar epithelial apoptosis, not counterbalanced by effective proliferation, has been proposed to be responsible for lung alveolar loss in both clinical and experimental studies (21, 36, 37). Although we are well aware that correlations do not imply a cause–effect relationship, the association between expression of IL-32 and degree of airflow limitation in our study supports the concept that the cytokine may be particularly relevant for the progression of the disease, regulating both inflammatory and apoptotic processes. Accumulating evidence points out that inflammation and apoptosis are indeed amplified in patients with advanced COPD (21, 38, 39), where one would expect only destructive lesions, thus setting the stage for the possibility of therapeutic interventions, even in end-stage disease.

We should acknowledge that ours is an observational study, and that we could not perform alternative methods to quantify IL-32 and TNF-{alpha} expression, such as ELISA or Western blot, nor a precise quantitative real-time PCR. Nevertheless, we believe that studies of this kind are important, because they have the potential to provide the clinical framework for proper functional investigations. Another potential bias in this study is that only a small number of subjects were examined in the control groups, but this is a common limitation because of the scarcity of patients without COPD who undergo a comprehensive clinical and functional characterization presurgically. Finally, a great proportion of subjects (either in COPD or in control groups) had lung cancer, and the presence of cancer itself may have influenced the results by enhancing inflammation (40). However, smokers with severe COPD who did not have lung cancer had the greatest levels of IL-32 expression. Therefore, if a bias due to cancer was present, this was in the opposite direction, and the up-regulation of IL-32 in COPD would be even greater if compared with better-matched control groups.

In conclusion, this study demonstrated overexpression of IL-32 in the lung periphery of smokers with COPD that was correlated with the presence of TNF-{alpha} and with the degree of airflow obstruction. Because increased levels of IL-32 were most prominent in alveolar macrophages, it may be worthwhile to investigate whether IL-32 is detectable with less invasive tools, such as bronchoalveolar lavage and induced sputum. If this is proven to be true, then IL-32 could be considered not solely as a therapeutic target in COPD, but also as a valid biomarker for disease progression.


   Acknowledgments
 
The authors thank Christina A. Drace for assistance in editing the manuscript.


   FOOTNOTES
 
Supported by University of Padova, Italian Ministry of University and Research; and an unrestricted grant from GlaxoSmithKline, UK.

This article has an online supplement, which is accessible from the issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200804-646OC on August 14, 2008

Conflict of Interest Statement: F.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.L.-O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.A.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.S. received lecture fees from GlaxoSmithKline, AstraZeneca, and Farmindustria, grants from Boehringer Ingelheim and GlaxoSmithKline, and reimbursements for participation in international conferences from GlaxoSmithKline, Boehringer Ingelheim, Merck, Sharpe & Dome, AstraZeneca, and Menarini.

Received in original form April 30, 2008; accepted in final form August 14, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lopez AD, Shibuya K, Rao C, Mathers CD, Hansell AL, Held LS, Schmid V, Buist S. Chronic obstructive pulmonary disease: current burden and future projections. Eur Respir J 2006;27:397–412.[Free Full Text]
  2. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C, et al.; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 2007;176:532–555.[Abstract/Free Full Text]
  3. Saetta M, Di Stefano A, Turato G, Facchini FM, Corbino L, Mapp CE, Maestrelli P, Ciaccia A, Fabbri LM. CD8+ T lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998;157:822–826.[Abstract/Free Full Text]
  4. Saetta M, Baraldo S, Corbino L, Turato G, Braccioni F, Rea F, Cavallesco G, Tropeano G, Mapp CE, Maestrelli P, et al. CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160:711–717.[Abstract/Free Full Text]
  5. Saetta M, Baraldo S, Turato G, Beghé B, Casoni GL, Bellettato CM, Rea F, Zuin R, Fabbri LM, Papi A. Increased proportion of CD8+ T-lymphocytes in the paratracheal lymph nodes of smokers with mild COPD. Sarcoidosis Vasc Diffuse Lung Dis 2003;20:28–32.[Medline]
  6. Gan WQ, Man SF, Senthilselvan A, Sin DD. Association between chronic obstructive pulmonary disease and systemic inflammation: a systematic review and a meta-analysis. Thorax 2004;59:574–580.[Abstract/Free Full Text]
  7. Saetta M, Mariani M, Panina-Bordignon P, Turato G, Buonsanti C, Baraldo S, Bellettato CM, Papi A, Corbetta L, Zuin R, et al. Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2002;165:1404–1409.[Abstract/Free Full Text]
  8. Grumelli S, Corry DB, Song LZ, Song L, Green L, Huh J, Hacken J, Espada R, Bag R, Lewis DE, et al. An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. PLoS Med 2004;1:e8.[CrossRef][Medline]
  9. Kim SH, Han SY, Azam T, Yoon DY, Dinarello CA. Interleukin-32: a cytokine and inducer of TNFalpha. Immunity 2005;22:131–142.[Medline]
  10. Dinarello CA, Kim SH. IL-32, a novel cytokine with a possible role in disease. Ann Rheum Dis 2006;65:iii61–iii64.[Abstract/Free Full Text]
  11. Nold MF, Nold-Petry CA, Pott GB, Zepp JA, Saavedra MT, Kim SH, Dinarello CA. Endogenous interleukin 32 controls cytokine and HIV-1 production. J Immunol 2008;181:557–565.[Abstract/Free Full Text]
  12. Goda C, Kanaji T, Kanaji S, Tanaka G, Arima K, Ohno S, Izuhara K. Involvement of IL-32 in activation-induced cell death in T cells. Int Immunol 2006;18:233–240.[Abstract/Free Full Text]
  13. Joosten LA, Netea MG, Kim SH, Yoon DY, Oppers-Walgreen B, Radstake TR, Barrera P, van de Loo FA, Dinarello CA, van den Berg WB. IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc Natl Acad Sci USA 2006;103:3298–3303.[Abstract/Free Full Text]
  14. Shioya M, Nishida A, Yagi Y, Ogawa A, Tsujikawa T, Kim-Mitsuyama S, Takayanagi A, Shimizu N, Fujiyama Y, Andoh A. Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease. Clin Exp Immunol 2007;149:480–486.[Medline]
  15. Calabrese F, Baraldo S, Bazzan E, Lunardi F, Sfriso P, Dinarello CA, Papi A, Zuin R, Saetta M. Overexpression of IL-32 in lung tissue from smokers with COPD. Am J Respir Crit Care Med 2007;175:A655.[CrossRef]
  16. Calabrese F, Baraldo S, Bazzan E, Lunardi F, Turato G, Lokar-Oliani K, Sfriso P, Rea F, Maestrelli P, Papi A, et al. IL-32, an emerging cytokine in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;177:A875.
  17. Saetta M, Di Stefano A, Maestrelli P, Turato G, Ruggieri MP, Roggeri A, Calcagni P, Mapp CE, Ciaccia A, Fabbri LM. Airway eosinophilia in chronic bronchitis during exacerbations. Am J Respir Crit Care Med 1994;150:1646–1652.[Abstract]
  18. Kim KH, Shim JH, Seo EH, Cho MC, Kang JW, Kim SH, Yu DY, Song EY, Lee HG, Sohn JH, et al. Interleukin-32 monoclonal antibodies for immunohistochemistry, Western Blot and ELISA. J Immunol Methods 2008;20:38–50.
  19. Maestrelli P, El Messlemani AH, De Fina O, Nowicki Y, Saetta M, Mapp C, Fabbri LM. Increased expression of heme oxygenase (HO)-1 in alveolar spaces and HO-2 in alveolar walls of smokers. Am J Respir Crit Care Med 2001;164:1508–1513.[Abstract/Free Full Text]
  20. Marian E, Baraldo S, Visentin A, Papi A, Saetta M, Fabbri LM, Maestrelli P. Up-regulated membrane and nuclear leukotriene B4 receptors in COPD. Chest 2006;129:1523–1530.[CrossRef][Medline]
  21. Calabrese F, Giacometti C, Beghe B, Rea F, Loy M, Zuin R, Marulli G, Baraldo S, Saetta M, Valente M. Marked alveolar apoptosis/proliferation imbalance in end-stage emphysema. Respir Res 2005;6:14.[CrossRef][Medline]
  22. Renda T, Baraldo S, Pelaia G, Bazzan E, Turato G, Papi A, Maestrelli P, Maselli R, Vatrella A, Fabbri LM, et al. Increased activation of p38 MAPK in chronic obstructive pulmonary disease. Eur Respir J 2008;31:62–69.[Abstract/Free Full Text]
  23. Aickin M, Gensler H. Adjusting for multiple testing when reporting research results: the Bonferroni vs Holm methods. Am J Public Health 1996;86:726–728.[Abstract/Free Full Text]
  24. Curtin F, Schulz P. Multiple correlations and Bonferroni's correction. Biol Psychiatry 1998;44:775–777.[CrossRef][Medline]
  25. Niewoehner DE, Klienerman J, Rice D. Pathologic changes in the peripheral airways of young cigarette smokers. N Engl J Med 1974;291:755–758.[Medline]
  26. Cosio MG, Hale KA, Niewoehner DE. Morphologic and morphometric effects of prolonged cigarette smoking on the small airways. Am Rev Respir Dis 1980;122:265–271.[Medline]
  27. Cosio MG, Majo J, Cosio MG. Inflammation of the airways and lung parenchyma in COPD: role of T cells. Chest 2002;121:160S–165S.[CrossRef][Medline]
  28. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004;350:2645–2653.[Abstract/Free Full Text]
  29. Agustí A, MacNee W, Donaldson K, Cosio M. Hypothesis: does COPD have an autoimmune component? Thorax 2003;58:832–834.[Free Full Text]
  30. Lee SH, Goswami S, Grudo A, Song LZ, Bandi V, Goodnight-White S, Green L, Hacken-Bitar J, Huh J, Bakaeen F, et al. Antielastin autoimmunity in tobacco smoking-induced emphysema. Nat Med 2007;13:567–569.[CrossRef][Medline]
  31. Feghali-Bostwick CA, Gadgil AS, Otterbein LE, Pilewski JM, Stoner MW, Csizmadia E, Zhang Y, Sciurba FC, Duncan SR. Autoantibodies in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;177:156–163.[Abstract/Free Full Text]
  32. Di Stefano A, Caramori G, Oates T, Capelli A, Lusuardi M, Gnemmi I, Ioli F, Chung KF, Donner CF, Barnes PJ, et al. Increased expression of nuclear factor-kappaB in bronchial biopsies from smokers and patients with COPD. Eur Respir J 2002;20:556–563.[Abstract/Free Full Text]
  33. Netea MG, Azam T, Lewis EC, Joosten LA, Wang M, Langenberg D, Meng X, Chan ED, Yoon DY, Ottenhoff T, et al. Mycobacterium tuberculosis induces interleukin-32 production through a caspase-1/IL-18/interferon-gamma–dependent mechanism. PLoS Med 2006;3:e277.[CrossRef][Medline]
  34. Atamas SP. Alternative splice variants of cytokines: making a list. Life Sci 1997;61:1105–1112.[CrossRef][Medline]
  35. Fletcher HA, Owiafe P, Jeffries D, Hill P, Rook GA, Zumla A, Doherty TM, Brookes RH; Vacsel Study Group. Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4delta2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulation. Immunology 2004;112:669–673.[CrossRef][Medline]
  36. Majo J, Ghezzo H, Cosio MG. Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema. Eur Respir J 2001;17:946–953.[Abstract/Free Full Text]
  37. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 2000;106:1311–1319.[Medline]
  38. Turato G, Zuin R, Miniati M, Baraldo S, Rea F, Beghé B, Monti S, Formichi B, Boschetto P, Harari S, et al. Airway inflammation in severe chronic obstructive pulmonary disease: relationship with lung function and radiologic emphysema. Am J Respir Crit Care Med 2002;166:105–110.[Abstract/Free Full Text]
  39. Retamales I, Elliott WM, Meshi B, Coxson HO, Pare PD, Sciurba FC, Rogers RM, Hayashi S, Hogg JC. Amplification of inflammation in emphysema and its association with latent adenoviral infection. Am J Respir Crit Care Med 2001;164:469–473.[Abstract/Free Full Text]
  40. Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001;357:539–545.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
P. J. Barnes
The Cytokine Network in Chronic Obstructive Pulmonary Disease
Am. J. Respir. Cell Mol. Biol., December 1, 2009; 41(6): 631 - 638.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
C. Sorrentino and E. Di Carlo
Expression of IL-32 in Human Lung Cancer Is Related to the Histotype and Metastatic Phenotype
Am. J. Respir. Crit. Care Med., October 15, 2009; 180(8): 769 - 779.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. A. Nold-Petry, M. F. Nold, J. A. Zepp, S.-H. Kim, N. F. Voelkel, and C. A. Dinarello
IL-32-dependent effects of IL-1{beta} on endothelial cell functions
PNAS, March 10, 2009; 106(10): 3883 - 3888.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200804-646OCv1
178/9/894    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Calabrese, F.
Right arrow Articles by Saetta, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Calabrese, F.
Right arrow Articles by Saetta, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2008 American Thoracic Society