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Published ahead of print on March 5, 2003, doi:10.1164/rccm.200205-487OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1695-1703, (2003)
© 2003 American Thoracic Society

Original Article

Expression of Macrophage Inflammatory Protein-3ß/CCL19 in Pulmonary Sarcoidosis

Agata Gibejova, Frantisek Mrazek, Daniela Subrtova, Veronika Sekerova, Jaroslava Szotkowska, Vitezslav Kolek, Roland M. du Bois and Martin Petrek

Departments of Immunology and Respiratory Medicine, Palacky University, Olomouc, Czech Republic; and Interstitial Lung Disease Unit, Royal Brompton Hospital, London, United Kingdom

Correspondence and requests for reprints should be addressed to Dr. Martin Petrek, M.D., Department of Immunology, Palacky University, I. P. Pavlova str. 6, Olomouc CZ-775 20, Czech Republic. E-mail: martinpe{at}email.cz


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, messenger RNA (mRNA) expression for novel T lymphocyte chemoattractants, leukotactin-1, macrophage inflammatory protein (MIP)-3{alpha} and MIP-3ß was investigated in bronchoalveolar lavage fluid (BALF) cells from patients with sarcoidosis, a T cell–mediated disease with typical CD4+ lymphocyte alveolitis. Of these three chemokines, only MIP-3ß mRNA was upregulated in sarcoidosis, and therefore, protein levels of this chemokine, its pharmacologic regulation, and association with disease clinical course were explored. MIP-3ß protein concentrations were elevated in BALF from sarcoid patients compared with control subjects (p = 0.001) and in patients with chest X-ray stage II chemokine protein levels were increased compared with stage I (p = 0.003). MIP-3ß protein was associated predominantly with alveolar macrophages and correlated with BALF lymphocytes and T cell subsets. mRNA expression for the MIP-3ß receptor, CC chemokine receptor 7, was increased in sarcoidosis and correlated with MIP-3ß protein levels. MIP-3ß mRNA and protein expression in BALF cells was suppressed by dexamethasone and cyclosporine A in vitro. In conclusion, MIP-3ß is implicated in T lymphocyte recruitment in sarcoidosis, is associated with disease progression, and is downregulated by drugs used for sarcoidosis treatment. This novel chemokine, therefore, represents a candidate for studies of sarcoidosis pathobiologic mechanisms.

Key Words: chemokine • leukotactin-1 • CC chemokine receptor 7 • dexamethasone • cyclosporine A

Sarcoidosis is a multiorgan granulomatous disorder most frequently affecting the lung that results from the accumulation of CD4+ T lymphocytes and macrophages (1). The mechanism of accumulation of inflammatory cells in the lung affected by sarcoidosis is not clear. However, proinflammatory cytokines and chemokines have been previously implicated in this process (2).

Chemotactic cytokines (chemokines) are low-molecular-weight cytokines traditionally divided into four subgroups (CC, CXC, CX3C, and C) based on the structure of N-terminal cysteine motifs; the current nomenclature in line with this division assigns serial numbers to individual chemokines (3). Chemokines regulate distribution of leukocytes and play an essential role in inflammation (4). Recently, a number of novel members of the chemokine superfamily have been described using bioinformatics (5), including the leukotactin-1 (Lkn-1)/CC chemokine ligand (CCL) 15/hemofiltrate CC chemokine-2 (6), macrophage inflammatory protein (MIP)-3{alpha}/CCL20/liver and activation-regulated chemokine (7), and MIP-3ß/CCL19/Epstein-Barr virus–induced molecule 1 ligand chemokine/Exodus 3/CKß-11 (7, 8).

The chemokine MIP-3ß is expressed especially in lymphoid tissues, whereas production of MIP-3{alpha} is found also in peripheral blood leukocytes and several fetal tissues (7, 8). Expression of Lkn-1 is observed in the liver, intestine, and lung leukocytes (9). The gene encoding chemokine MIP-3ß maps on human chromosome 9, whereas most other CC chemokine genes (including MIP-3{alpha} and Lkn-1) are clustered on chromosome 17 (8). Although there are significant differences in tissue distribution of Lkn-1, MIP-3{alpha}, and MIP-3ß, mRNA transcripts for all three chemokines have been previously found in samples of human lung tissue (7, 9, 10).

The chemokine MIP-3ß is chemoattractant for T and B lymphocytes (11, 12), dendritic cells (13), macrophage progenitor cells (14), and natural killer cells (15). It might, therefore, play an important role in the trafficking of T cells in the thymus and migration of T and B cells to secondary lymphoid organs (12, 16). Furthermore, MIP-3ß has been recently shown to mediate rapid adhesion of naive CD4+ T lymphocytes to activated endothelial cells supporting the role of this chemokine in regulation of lymphocyte homing (17). MIP-3ß acts through CC chemokine receptor 7 (CCR7) (8). The chemokines Lkn-1 and MIP-3{alpha} are also lymphocyte attractants, although compared with MIP-3ß, their promigratory effect on lymphocytes is less intensive and their chemotactic activity is more promiscuous. Lkn-1 attracts monocytes, lymphocytes, and eosinophils via the chemokine receptors CCR1 and CCR3 (6, 18). MIP-3{alpha} exhibits promigratory effects on lymphocytes, neutrophils, and immature dendritic cells via the CCR6 receptor (13, 19).

Regarding their promigratory effect on lymphocytes, we hypothesized that novel lymphocyte attractant chemokines MIP-3ß and also Lkn-1, and MIP-3{alpha} may contribute to the development of the CD4+ lymphocyte alveolitis, which accompanies pulmonary sarcoidosis. We have, therefore, investigated the mRNA expression of these three chemokines in bronchoalveolar lavage fluid (BALF) cells from patients with sarcoidosis in comparison with cells from healthy subjects and have also examined the relationship between chemokine mRNA expression and BALF cellular profile. These expression studies revealed upregulation of MIP-3ß transcripts, which was associated with lymphocyte alveolitis. We have, therefore, focused on MIP-3ß. Protein levels of MIP-3ß were measured in BALF of patients in comparison to control subjects and their relationship to clinical course of sarcoidosis, as assessed by chest X-ray stage and need for treatment, was evaluated. Furthermore, cell-associated MIP-3ß protein in BALF was identified by immunocytochemistry, and mRNA expression of MIP-3ß receptor, CC chemokine receptor (CCR) 7, was determined in BALF cells. Finally, to explore how current therapeutic approaches to sarcoidosis interfere with MIP-3ß chemokine expression, we have studied the effects of dexamethasone and cyclosporine A on MIP-3ß mRNA and protein expression in vitro. Some of the results of these studies have been previously reported in the form of an abstract (20).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Population
Bronchoalveolar lavage was performed according to a standard procedure in 78 patients with sarcoidosis and 11 control subjects. The control group consisted of subjects who at the time of presentation and subsequently showed no clinical signs of lung inflammation; they had no lung disease in their medical history. All had normal BALF cytology, immunology, and microbiology.

The diagnosis of pulmonary sarcoidosis was based on typical clinical features together with granulomas on lung biopsies and supported by the BALF cellular profile and was compatible with the criteria contained in the International Statement on Sarcoidosis (21). Regarding chest radiography, patients were divided into stages (stage 0: n = 2, stage I: n = 48, stage II: n = 25, and stage III: n = 3). An additional subdivision of patients was established to provide an index of disease course: patients requiring corticosteroid treatment (n = 40) and patients in whom treatment was not necessary (i.e., the disease resolved spontaneously) (n = 38). No patient received corticosteroid treatment before bronchoalveolar lavage. The treatment scheme did not differ from that recommended in the International Statement on Sarcoidosis (21). Treatment with steroids was indicated according to an accepted protocol: (1) all patients with chest X-ray stage III disease at presentation, (2) patients with progressing and/or symptomatic stage II disease, and (3) patients with persistent stage I or II. Patients with persistent disease were treated after at least 6 months of disease observation. Detailed clinical characterization of the study groups is shown in Table 1 . The study was performed with the approval of the Ethics Committee of the Medical Faculty of Palacky University Olomouc.


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  		TABLE 1. Clinical and laboratory data of investigated subjects

 
Semiquantification of Lkn-1, MIP-3{alpha}, MIP-3ß, and CCR7 mRNA Expression in Bronchoalveolar Lavage Cells by Reverse Transcription-Polymerase Chain Reaction
The methods used for isolation of mRNA from unseparated BALF cells and for semiquantification of chemokine mRNA expression are described elsewhere (22, 23). Chemokine and receptor-specific polymerase chain reaction were performed using modified amplification protocols (24, 25); primer sequences are shown in Table 2 . Authenticity of MIP-3ß amplification was confirmed by direct sequencing (data not shown). ß-Actin–specific polymerase chain reaction has been described elsewhere (23).


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  		TABLE 2. Characterization of oligonucleotide sequences of the primers used for specific polymerase chain reaction

 
Optical densities of amplicons were determined using the software Quantiscan (Biosoft, Ferguson, MO). mRNA expression was normalized to the expression of the ß-actin gene as the optical density ratio (ODR). This approach (description in detail in Petrek [23]) has been previously validated in mRNA semiquantification of other chemokines (26).

MIP-3ß ELISA
MIP-3ß protein levels were measured in BALF and cultured supernatants by specific DuoSet ELISA Development kit (R&D Systems, Abingdon, UK) according to the manufacturer's instructions. The detection limit of this assay was 4 pg/ml. The method is described in detail in the online supplement (section A).

Immunocytochemistry
MIP-3ß protein was detected on cytocentrifuge preparations of BALF cells using the streptavidin-biotin/horseradish peroxidase method with anti–MIP-3ß monoclonal antibody (R&D Systems, Abingdon, UK). The method is described in detail in the online supplement (section B).

Effect of Immunomodulators: In Vitro Regulation Experiments
Bronchoalveolar cells were cultured (106/ml cells) in RPMI-1640 medium (ÚSOL, Prague, Czech Republic) supplemented with 8% fetal calf serum (Flow Labs, Irvine, UK) and 10 ng/ml of tumor necrosis factor-{alpha} (NIBSC, Potters Bar, UK) in 5% CO2 atmosphere at 37°C. Three types of cultures were set up: cells cultured alone and cells cultured in the presence of dexamethasone or cyclosporine A (dexamethasone acetate, 10-6 M [Léciva, Prague, Czech Republic]; and cyclosporine A, 10 ng/ml [Sandimmune, Sandoz, Switzerland]). After a 21-hour culture, the supernatants were used for determination of protein by ELISA, and the cells were used for mRNA extraction for subsequent reverse transcription-polymerase chain reaction experiments.

Statistical Analysis
The data are reported as median with first to third quartile (interquartile range [IQR]). Comparisons of mRNA and protein expression between study groups were performed using the nonparametric Mann-Whitney U-test. Spearman's rank correlation was used to assess the relationships between chemokine expression and cellular profile of BALF and the clinical course of sarcoidosis. Paired t test analysis was used to investigate differences in expression in vitro culture experiments. Differences with a p value of less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lkn-1, MIP-3{alpha}, and MIP-3ß mRNA Expression in BALF Cells
To investigate chemokine mRNA expression in patients with sarcoidosis and control subjects, mRNA extracted from unseparated BALF cells by biomagnet separation was reverse transcribed to cDNA and specific polymerase chain reaction for the chemokine Lkn-1, MIP-3{alpha}, and MIP-3ß genes were performed. The levels of mRNA expression (ODR) were determined for each individual by normalizing to the expression of the ß-actin gene.

Lkn-1 mRNA transcripts were found in similar frequency in 25 of 30 (83%) of patients with sarcoidosis and in 11 of 11 (100%) of control subjects. Lkn-1 mRNA expression was similar in both groups tested (ODR, median [IQR]; control subjects, 0.66 [0.38–0.93]; sarcoidosis, 0.62 [0.25–0.85]; p = 0.814; Figure 1A) .



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  		Figure 1. Messenger RNA (mRNA) expression of the chemokine leukotactin (Lkn)-1 (A) and macrophage inflammatory protein (MIP)-3{alpha} (B) in bronchoalveolar lavage fluid (BALF) cells obtained from control subjects (n = 11) and patients with sarcoidosis (n = 30). mRNA expression is semiquantified using an ODR (group medians are indicated by horizontal bars).

 
MIP-3{alpha} mRNA transcripts were observed in similar frequency in 28 of 30 (93%) of patients with sarcoidosis and in 10 of 11 (91%) of control subjects. MIP-3{alpha} mRNA expression in BALF cells from patients with sarcoidosis and from control subjects was similar (ODR, median [IQR]; control subjects, 0.57 [0.26–0.95]; sarcoidosis, 0.62 [0.42–0.91]; p = 0.659; Figure 1B).

MIP-3ß mRNA was detected in 70% (21 of 30) of patients with sarcoidosis but only in 55% (6 of 11) of control subjects. Importantly, MIP-3ß mRNA expression was increased in patients with sarcoidosis in comparison with control subjects (ODR, median [IQR]; control subjects, 0.00 [0.00–0.17]; sarcoidosis, 0.33 [0.00–0.47]; p = 0.035; Figure 2A) .



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  		Figure 2. (A) mRNA expression of the chemokine MIP-3ß in BALF cells obtained from control subjects (n = 11) and patients with sarcoidosis (n = 30). mRNA expression is semiquantified using an optical density ratio (ODR) (group medians are indicated by horizontal bars). (B) Relationship of MIP-3ß mRNA expression (ODR MIP-3ß/ß-actin) with BALF CD4+/CD8+ T cell ratio. MIP-3ß mRNA expression versus BALF CD4+/CD8+ T cell ratio (rs = 0.541, p < 0.001). Open triangles = control subjects; closed circles = subjects with sarcoidosis.

 
Relationship between Lkn-1, MIP-3{alpha}, and MIP-3ß mRNA Expression and BALF Cells
Because Lkn-1, MIP-3{alpha}, and MIP-3ß are novel lymphocyte attractant chemokines, we investigated a possible relationship of their mRNA expression with BALF lymphocyte numbers. mRNA expression of Lkn-1 and MIP-3{alpha} did not correlate with the number of BALF lymphocytes nor with the CD4+/CD8+ T cell ratio and BALF lymphocyte subsets. MIP-3ß mRNA expression correlated with absolute (rs = 0.389, p = 0.017) and relative (rs = 0.373, p = 0.019) BALF lymphocyte count and moreover with CD4+ lymphocytes (rs = 0.488, p = 0.003) but not with the CD8+ subset. Furthermore, there was a striking association between MIP-3ß mRNA expression and BALF CD4+/CD8+ T cell ratio (rs = 0.541, p < 0.001; Figure 2B). The relationship between Lkn-1, MIP-3{alpha}, and MIP-3ß mRNA expression and number of BALF macrophages, neutrophils, and eosinophils was also investigated, but no association was observed (p > 0.05).

MIP-3ß Protein Levels in BALF and Their Relationship to BALF Cell Profile
Because of the finding of upregulated mRNA for MIP-3ß and its association with numbers of total BALF lymphocytes, we explored this chemokine also at the protein level. BALF protein concentrations were significantly increased in patients with sarcoidosis in comparison to control subjects (median [IQR], pg/ml; control subjects, 5.9 [5.2–7.0]; sarcoidosis, 11.2 [6.9–47.6]; p = 0.001; Figure 3A) . MIP-3ß BALF protein levels correlated with mRNA expression (rs = 0.320, p = 0.036). The MIP-3ß BALF protein levels strongly correlated with absolute (rs = 0.500, p < 0.001; Figure 3B) and relative (rs = 0.53, p < 0.001) numbers of total BALF lymphocytes. There was a strong correlation between MIP-3ß BALF protein concentrations and absolute number of both CD4+ and the CD8+ BALF lymphocyte subsets (rs = 0.370, p = 0.002 and rs = 0.340, p = 0.005, respectively). The relationship between MIP-3ß protein expression and number of BALF macrophages, neutrophils, and eosinophils was also investigated, but no association was observed (p > 0.05).



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  		Figure 3. (A) Concentrations of MIP-3ß protein (pg/ml, expressed by log) in BALF obtained from control subjects (n = 11) and patients with sarcoidosis (n = 78) (group medians are indicated by horizontal bars). (B) Relationship of MIP-3ß protein concentrations (pg/ml, expressed by log) with BALF lymphocytes. MIP-3ß protein versus BALF absolute lymphocyte number (rs = 0.500, p < 0.001). Open triangles = control subjects; closed diamonds = subjects with sarcoidosis.

 
MIP-3ß Protein Levels in BALF and Clinical Course of Sarcoidosis
To evaluate whether MIP-3ß is associated with the course of sarcoidosis, its protein expression was analyzed in subgroups of patients with different chest X-ray stage and distinct requirement for treatment. MIP-3ß concentrations were significantly elevated in patients with chest x-ray stage II (n = 25) in comparison to patients with stage I (n = 48) (median [IQR], pg/ml; stage I, 9.6 [6.4–24.2]; stage II, 48.6 [9.3–213.0]; p = 0.003; Figure 4A) . The BALF protein levels of MIP-3ß were higher in patients requiring treatment (n = 40, median [IQR], pg/ml; 16.9 [8.5–106.8]) than in patients with spontaneous remission (n = 38, median [IQR], pg/ml; 10.1 [6.5–38.9]; Figure 4B); the difference did not, however, attain significance (p = 0.07).



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  		Figure 4. Concentrations of MIP-3ß protein (pg/ml, expressed by log) in BALF obtained from patients with different chest X-ray stage (A) and distinct disease course (B) (group medians are indicated by horizontal bars).

 
Determination of Cell-associated MIP-3ß Protein by Immunocytochemistry
To identify the cellular source of MIP-3ß, cytocentrifuge preparations of BALF cells obtained from 17 patients with sarcoidosis and from 6 control subjects were immunostained for MIP-3ß protein. The protein was found to be expressed in all investigated patients and control samples. Strong, uniform expression of MIP-3ß was observed in alveolar macrophages (Figure 5) . MIP-3ß protein was associated also with lymphocytes; however, its expression was limited to approximately one-third of BALF lymphocytes, and it was less intensive than in the case of macrophage-associated MIP-3ß. Semiquantitative analysis of MIP-3ß–immunostained preparations revealed a trend to higher proportion of MIP-3ß strongly positive macrophages in patients with sarcoidosis in comparison to control subjects (see Figure E1 in the online supplement).




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  		Figure 5. Detection of MIP-3ß protein in BALF cells from patients with sarcoidosis; representative result of immunocytochemistry experiments. Positively stained cells were observed in BALF cytospin preparations incubated with anti–MIP-3ß antibody (A), but not with the irrelevant control antibody (B). MIP-3ß was localized predominantly to the cytoplasm of macrophages (black arrows); less intensive staining was associated also with a minor proportion of BALF lymphocytes (white arrows) (streptavidin-biotin/horseradish peroxidase method with 3,9 amino-ethyl-carbazole as substrate and counterstained with hematoxylin; original magnification x125).

 
CCR7 mRNA Expression in BALF Cells and Its Relationship to BALF Cell Profile
Because MIP-3ß is a ligand for CCR7, we have attempted to analyze mRNA expression of this receptor molecule. CCR7 mRNA was detected in 67% (20 of 30) of patients affected by sarcoidosis and only in 36% (4 of 11) of control subjects. Patients with sarcoidosis had higher CCR7 mRNA expression (ODR, median [IQR]; sarcoidosis, 0.25 [0.00–0.51]) than control subjects (ODR, median [IQR]; control subjects, 0.00 [0.00–0.21]; Figure 6A) ; the difference did not, however, attain significance (p = 0.066).



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  		Figure 6. (A) mRNA expression of the chemokine receptor CCR7 in BALF cells obtained from control subjects (n = 11) and patients with sarcoidosis (n = 30). mRNA expression is semiquantified using an ODR (group medians are indicated by horizontal bars). (B) Relationship of CCR7 mRNA expression (ODR CCR7/ß-actin) with BALF CD4+/CD8+ T cell ratio. CCR7 mRNA expression versus BALF CD4+/CD8+ T cell ratio (rs = 0.435, p = 0.006). Open triangles = control subjects; closed circles = subjects with sarcoidosis.

 
CCR7 mRNA expression correlated with absolute (rs = 0.356, p = 0.031) and relative (rs = 0.338, p = 0.035) numbers of total BALF lymphocytes. It also strongly associated with CD4+ lymphocytes (absolute number, rs = 0.469, p = 0.005; relative number, rs = 0.380, p = 0.019) but not with CD8+ T cell numbers. Furthermore, there was a relationship between CCR7 mRNA expression and BALF CD4+/CD8+ T cell ratio (rs = 0.435, p = 0.006; Figure 6B). The association between CCR7 mRNA expression and number of other BALF cells was also investigated: There was a relationship between CCR7 mRNA and absolute (rs = 0.422, p = 0.009) and relative (rs = 0.369, p = 0.021) number of BALF neutrophils. Interestingly, CCR7 mRNA expression correlated with MIP-3ß BALF protein levels (rs = 0.314, p = 0.046).

Effect of Immunomodulators on MIP-3ß mRNA and Protein Expression In Vitro
In vitro experiments investigating the effects of dexamethasone and cyclosporine A on MIP-3ß mRNA and protein expressions were performed to explore the modulatory effect of these drugs on chemokine expression. The results are summarized in Figures 7A and 7B , respectively. Tumor necrosis factor-{alpha}–induced MIP-3ß expression was significantly suppressed in cells cultured in the presence of dexamethasone and cyclosporine A. The levels of mRNA were reduced in 10 of 12 (83%) cultures treated by dexamethasone and in 7 of 8 (88%) cultures in the presence of cyclosporine A (dexamethasone, p = 0.004; cyclosporine A, p = 0.009). MIP-3ß protein concentrations were downregulated in 9 from 12 (75%) cultures treated by dexamethasone and in 6 from 8 (75%) cultures affected by cyclosporine A (dexamethasone, p = 0.020; cyclosporine A, p = 0.029).



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  		Figure 7. (A) Effect of dexamethasone (Dx) on MIP-3ß mRNA and protein expression in vitro. The effect is expressed as an index of modulation that normalizes the data to the tumor necrosis factor (TNF)-stimulated values for mRNA and protein expression in 12 cultures treated with Dx (mean ± SEM). Paired t test was used to investigate differences in MIP-3ß expression in modulated cultures in vitro. TNF-{alpha} induced MIP-3ß protein expression in cultures treated by Dx versus cultures without effect of Dx (p = 0.020). TNF-{alpha} induced MIP-3ß mRNA expression in cultures treated by Dx versus cultures without effect of Dx (p = 0.004). (B) Effect of cyclosporine A (CyA) on MIP-3ß mRNA and protein expression in vitro. The effect is expressed as an index of modulation that normalizes the data to the TNF-stimulated values for mRNA and protein expression in 8 cultures treated with CyA (mean ± SEM). Paired t test was used to investigate differences in MIP-3ß expression in modulated cultures in vitro. TNF-{alpha} induced MIP-3ß protein expression in cultures treated by CyA versus cultures without effect of CyA (p = 0.029). TNF-{alpha} induced MIP-3ß mRNA expression in cultures treated by CyA versus cultures without effect of CyA (p = 0.009).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The accumulation of immune cells (macrophages and CD4+ T lymphocytes) in the pulmonary interstitium and alveoli is a typical feature of patients with lung inflammation in sarcoidosis (1). Several chemokines, such as regulated upon activation, normal T cell expressed and secreted (CCL5), MIP-1{alpha} (CCL3), MIP-1ß (CCL4), monocyte chemoattractant protein-1 (CCL2), and interferon-{gamma} inducible protein-10 (CXCL10), have already been implicated in the recruitment of leukocytes to the lungs of these patients (27–30). In this study, we have focused on novel candidate mediators of this migration process: lymphocyte attractant chemokines Lkn-1, MIP-3{alpha}, and MIP-3ß, which were recently identified by bioinformatics (5). Of these three novel chemokines, MIP-3ß expression was upregulated in BALF cells and also in BALF fluid from patients with sarcoidosis by comparison with control subjects. Importantly, MIP-3ß BALF protein levels paralleled the disease course and were associated with the extent of CD4+ T lymphocyte alveolitis. Finally, studies directed at MIP-3ß receptor, CCR7, suggested an increase of CCR7 mRNA expression in BALF cells from sarcoid patients.

The findings of this study are in compliance with our initial hypothesis that the novel chemokines, including MIP-3ß, may contribute to the recruitment of lymphocytes to the sarcoid lung. First, both MIP-3ß mRNA and protein were upregulated in BALF from patients with sarcoidosis. Second, there was a strong correlation between MIP-3ß expression and total BALF lymphocytes as well as with the absolute BALF CD4+ T cell count. Furthermore, our immunocytochemistry experiments revealed that although virtually all alveolar macrophages were immunostained for MIP-3ß protein, only minor MIP-3ß expression was associated with some BALF lymphocytes. Semiquantitative analysis showed more intensive immunostaining of sarcoid macrophages compared with macrophages from our control subjects, indicating that in disease there is more MIP-3ß per cell. In this context, the possibility that increased chemokine detected in sarcoid macrophages arose from phagocytosis of external chemokine is unlikely. Other than macrophages, there are only minor producers of MIP-3ß present in BALF. Dendritic cells, which also express this chemokine, account for no more than 1% of the cells in the bronchoalveolar compartment (31), and interestingly, their typical phenotypic markers can be acquired by sarcoid alveolar macrophages (32). All of these facts imply that alveolar macrophages are indeed the major source of BALF MIP-3ß protein, which is also concordant with reports of MIP-3ß cellular expression in other tissues (7, 33). However, a definitive answer about the source of the chemokine would require evaluation of transcripts at a single-cell level. Finally, we report here that there is a relationship between the expression of MIP-3ß and its receptor molecule, CCR7: The number of CCR7 transcripts correlated with MIP-3ß expression, and in patients with sarcoidosis, there was a marked trend to an increase of mRNA expression for the CCR7 molecule, which is, however, not fully specific for MIP-3ß, as it may bind another ligand, chemokine secondary lymphoid tissue chemokine (CCL21) (34).

Taken together, our findings are in favor of the explanation that macrophage-derived MIP-3ß contributes to the development of alveolitis in sarcoid lung by promoting CD4+ lymphocyte recruitment. There are functional data on preferential attraction of CD4+ T cells by MIP-3ß (11). However, a definite role for this chemokine in the cell-migration process in sarcoidosis cannot be assigned before chemotaxis experiments with BALF cells are performed.

In contrast to MIP-3ß, mRNA expression of the other two chemokines, Lkn-1 and MIP-3{alpha}, did not differ between patients and control subjects, and their expression was not related to the extent of lymphocyte alveolitis. Rather than acting as disease mediators, these chemokines may contribute to immune surveillance by participation in regulation of physiological T cell trafficking (16, 35). Although mRNA for these two chemokines was expressed in the great majority of study subjects, MIP-3ß expression was much more heterogeneous. Apart from many negative control subjects, no MIP-3ß transcripts were detected in approximately one-third of patients. We are not aware of any method confounder: The BALF procedure and its processing were standardized as in our previous investigations (27, 29), and our mRNA isolation procedure and semiquantitative reverse transcription-polymerase chain reaction method were carefully validated with regard to the specific conditions of this study (22, 23). In protein experiments, free immunoreactive MIP-3ß was detected in all study subjects, but the distribution of individual values of BALF chemokine concentrations resembled the pattern of the mRNA data; importantly, mRNA level and protein concentrations correlated. It is indeed possible that the commercial ELISA assay was more sensitive and detectable, albeit low protein levels were determined also in the samples in which no MIP-3ß transcripts could be identified. The observed heterogeneity of MIP-3ß expression may be due to interindividual differences in the hierarchy of chemokine expression. Microarray technology could be used for future assessment of parallel expression of a set of chemokines and also for the analysis of the contribution of gene polymorphisms to differences in interindividual expression.

As already mentioned, upregulated expression of several CC and CXC chemokines has been reported before in sarcoidosis (36), and a relationship between monocyte chemoattractant protein-1 (CCL2), MIP-1{alpha} (CCL3), and MIP-1ß (CCL4) protein levels and clinical course of disease has been described (28, 29, 37). In this context, it is interesting that the upregulation of MIP-3ß protein was observed in the BALF of our patients affected by chest X-ray stage II in comparison to patients with stage I. There was also a difference in MIP-3ß expression in patients subdivided according to the need for corticosteroid treatment (as a measure of disease evolution): MIP-3ß protein levels tended to be higher in patients requiring treatment than in patients with spontaneous remission. The lack of a significant difference between active and inactive disease may be due to the fact that the relatively small number of patients may have been insufficient to detect a difference in MIP-3ß in patients with active disease. The treatment scheme did not differ from that recommended in the International Statement on Sarcoidosis (21), and therefore, it is less likely that lack of significance is caused by usage of different criteria for starting steroids by physicians involved in this study. Further studies based on additional functional and clinical characteristics including long-term patient follow-up are, however, necessary for a more definitive interpretation of the clinical significance of chemokine upregulation.

From a practical point of view, it is important that drugs used for sarcoidosis treatment, dexamethasone and cyclosporine A, suppressed in vitro MIP-3ß expression in BALF cells from patients with sarcoidosis. By analogy, our data have an in vivo correlate in the clinical observation of Hashimoto and coworkers (37): After successful corticosteroid treatment of patients with sarcoidosis, the elevated serum levels of other chemokines (monocyte chemoattractant protein-1 and MIP-1{alpha}) returned to normal values. The potential practical importance of the reported elevation of MIP-3ß in sarcoidosis, including data on its pharmacologic regulation, would be strengthened by other in vivo data. However, we did not find any report of work in animal model with this chemokine that could be used to support or dispute its potential as a therapeutic target. Also, published information on in vivo expression of MIP-3ß and other investigated chemokines is limited. To our knowledge, MIP-3ß expression in human disease has so far been restricted to Sjögren's syndrome (38) and atherosclerosis (33), in which also Lkn-1 was detected (39). Reports of disease expression of MIP-3{alpha} are more frequent (40–42), but there has been no report of its expression in lung diseases. The aforementioned reports also imply the novelty of our data, which means that their significance has to be verified in future independent studies.

In conclusion, we report here for the first time that the chemokine MIP-3ß and its CCR7 (but not chemokines Lkn-1 and MIP-3{alpha}) are upregulated in the bronchoalveolar fluid cells from patients with sarcoidosis. Furthermore, we demonstrate that MIP-3ß mRNA and protein levels and CCR7 mRNA expression correlate with the number of lymphocytes in the bronchoalveolar compartment and that MIP-3ß protein was localized mainly in alveolar macrophages. These findings indicate that MIP-3ß is implicated in the complex network between lymphocytes and cytokines, which sets the stage for the subsequent development of sarcoid granuloma. Finally, we have shown that MIP-3ß expression can be suppressed by dexamethasone and cyclosporine A at both the mRNA and protein level. These data and our observation of a relationship between MIP-3ß protein and the clinical course of disease suggest that this chemokine may be an important mediator in the pathobiologic mechanisms of sarcoidosis.


   Acknowledgments
 
Determination of lymphocyte subsets in BALF was performed in part by Dr. M. Ordeltova. Dr. J. Drabek designed primer pairs for Lkn-1 and MIP-3{alpha}. The authors gratefully acknowledge technical assistance from Ms. A. Vevodova and the staff of Bronchology Unit, Department of Respiratory Medicine, Faculty Hospital Olomouc.


   FOOTNOTES
 
Supported by the Czech Ministry of Health (IGA grant no. 3768–3 to M.P.); partial funding from the Czech government fund (MSMT J14/98.151100002) and also from a Travel Fellowship granted to M.P. by the British Society for Immunology.

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

Received in original form May 30, 2002; accepted in final form March 4, 2003


   REFERENCES
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 RESULTS
 DISCUSSION
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