Bone Marrow-Derived Progenitor Cells in End-Stage Lung Disease
Bone Marrow-Derived Progenitor Cells in End-Stage Lung Disease
Circulating bone marrow-derived cell populations were defined by (1) Clara Cell Secretory Protein (CCSP) expression for epithelial-like progenitors and by (2) dual expression of CD45 and intracellular collagen-1 expression for fibrocytes. To validate the expression of CCSP mRNA in the bone marrow and peripheral blood, Taqman PCR was utilized. CCSP mRNA was detected in human bone marrow cells (BMCs) and peripheral blood mononuclear cells (PBMCs) from 3 randomly selected lung transplant recipients, as well as in control lung bronchus tissue, but absent in experimental controls (no reverse transcription and no template controls) (Figure 1A-B). As a further proof-of-principle, peripheral blood cells from a healthy volunteer were isolated and sorted for CCSP by flow cytometery, representing less than 1% of total PBMCs, and then analyzed by PCR. CCSP mRNA was also detected in the pre-sorted population but not in CCSP-negative sorted cells (Figure 1C). The resulting amplification product was further analyzed by gel electrophoresis to confirm the correct amplicon size (Figure 1D), and compared to positive human bronchus tissue mRNA and a mixture of sample mRNAs not subjected to reverse transcription (NRT) to control for genomic DNA contamination. All subsequent quantification of progenitor cells populations was determined by flow cytometry. A representative plot identifying the positively gated populations based on initial isotype staining of 1% is shown for both CCSP cells and CD45Collagen-1 fibrocytes (Figure 1E-F). Further details of the gating strategy have been previously published.
(Enlarge Image)
Figure 1.
Identification of progenitor populations. Taqman PCR measurement of (A) Clara Cell Secretory Protein (CCSP) gene expression in human bone marrow cells (BMCs), (B) peripheral blood mononuclear cells (PBMCs), and (C) PBMCs pre-sorted for CCSP by flow cytometry. No template and no reverse transcription negative controls are included. (D) Gel electrophoresis of PCR product after Taqman-based amplification. Positive control (bronchus) and negative no reverse transcription (NRT) controls are included. (E) Flow cytometry gating for measurement of CCSP PBMCs based on isotype control staining. (F) Flow cytometry gating for measurement of CD45Collagen-1 peripheral blood leukocytes (PBLs) based on isotype control staining.
Bone marrow and peripheral blood samples were collected from patients at the time of lung transplant (n = 154). A summary of relevant lung recipient demographics, including age, gender, diagnosis, diabetes status, BMI, and graft number is presented in Table 1. In addition, blood and bone marrow samples were obtained from lung donors prior to organ recovery (n = 36), the details of which are summarised in Table 2. For logistical reasons, not every patient undergoing lung transplantation or donor procurement could be analyzed in this study. A comparison of recipient and donor demographics from patients included in this analysis compared to the demographics of all those transplanted at our centre in the same time period identified no significant differences in these parameters (Additional file 2: Table S1 and Additional file 3: Table S2).
No significant relationships were identified when these progenitor cell populations were analysed by age, gender, or BMI (data not shown). There were significant differences in the proportion of bone marrow-derived cells populations based on underlying lung disease. An increase in the proportion of CCSP cells was found in the bone marrow of CF patients when compared to lung donors (CF median = 1.33%, Donor median = 0.98, p < 0.05) (Figure 2A). We chose to use lung transplant donors as a comparison group as we had access to sternal marrow biopsy material obtained using identical collection techniques, not easily obtained in any other way. Different cell proportions were also found for CCSP cells in the peripheral blood. Specifically, CF patients had a greater proportion of CCSP cells when compared to lung donors (2.28% CF vs. 1.90% Donor, p < 0.05) (Figure 1B), while BO patients had a significantly lower proportion of CCSP PBMCs compared to donors (0.55% vs. 1.90% Donor, p < 0.05). When circulating CD45collagen-1 fibrocytes were compared between disease groups, an increased proportion was found in both BO patients (p < 0.001) and PF (p < 0.05) patients, when compared to Donors (Median BO = 7.02%, Median PF = 2.07%, Median Donors = 0.85%) (Figure 2C). As the changes in cell numbers appeared to reciprocal, the ratio of fibrocytes-to-CCSP PBMCs was calculated, and a similar pattern was found, where a predominantly fibrotic phenotype is represented in BO and PF, but not in other lung pathologies (Figure 2D).
(Enlarge Image)
Figure 2.
Differential progenitor cell profiles in end-stage lung disease patients. (A) Percentage of bone marrow cells (BMCs) positive for CCSP in each disease group (n = 26 donor, n = 5 Bronchiolitis Obliterans (BO), n = 27 Cystic Fibrosis (CF), n = 34 Chronic Obstructive Pulmonary Disease (COPD), n = 41 Pulmonary Fibrosis (PF), n = 11 Pulmonary Hypertension (PH)). (B) Percentage of peripheral blood mononuclear cells (PBMCs) positive for CCSP in each disease group. (n = 29 donor, n = 6 BO, n = 33 CF, n = 41 COPD, n = 53 PF, n = 13 PH). (C) Percentage of peripheral blood leukocytes (PBLs) positive for CD45 and collagen-1 in each disease group (n = 17 donor, n = 6 BO, n = 14 CF, n = 22 COPD, n = 26 PF, n = 8 PH). (D) Ratio of CCSP PBMCs to CD45collagen-1 fibrocytes in each disease group, compared to lung donors (n = 13 donor, n = 6 BO, n = 10 CF, n = 17 COPD, n = 18 PF, n = 8 PH). Kruskal-Wallis test with Dunn's multiple comparison post-hoc analysis. Boxes show the median, 25th and 75th percentiles. Whiskers represent the 2.5 and 97.5 percentiles.
In order to determine if the proportion of CCSP cells was reflective of the total number of CCSP cells, the absolute cell numbers were determined using total leukocyte counts collected from clinical data (10/L). When absolute cell numbers were compared, the differences in progenitor cell numbers between disease groups were still statistically significant (Additional file 4: Figure S1).
To investigate the relationship between CCSP cells within the bone marrow and the proportion in the peripheral blood, data was analyzed including all disease groups together. A significant correlation was found between the number of bone marrow and peripheral blood CCSP cells (Figure 3A). In contrast, no relationship was found between the number of fibrocytes and either CCSP BMC or PBMCs (Figure 3B-C).
(Enlarge Image)
Figure 3.
Relationship between progenitor cell populations. (A) Correlation between the percentage of Clara Cell Secretory Protein (CCSP) Peripheral Blood Mononuclear Cells (PBMCs) and CCSP Bone Marrow Cells (BMCs) (n = 119 pairs). (B) Lack of correlation between the percentage of CD45Collagen-1 fibrocytes and CCSP BMCs (n = 59 pairs). (C) Lack of correlation between the percentage of CD45Collagen-1 fibrocytes and CCSP PBMC (n = 74 pairs). Spearman rank test with correlation coefficient.
Analysis of clinical disease indicators relative to progenitor cell numbers was done using spirometric lung function values, using the ratio of the forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) (FEV1/FVC) ratio for CF and COPD patients, or based on the percentage of predicted FVC for PF patients. No direct relationships were found between lung function measurements when compared with the total number of epithelial-like progenitors in the bone marrow or peripheral blood or with circulating fibrocytes (Figure 4A-I).
(Enlarge Image)
Figure 4.
Lung function and progenitor cell profiles. No correlation in Cystic Fibrosis (CF) patients between FEV1/FVC and (A) Clara Cell Secretory Protein (CCSP+) bone marrow cells (BMCs) (n = 27), (B) CCSP + peripheral blood mononuclear cells (PBMCs) (n = 32), or (C) CD45 + Collagen-1+ fibrocytes (n = 14). No correlation in Chronic Obstructive Pulmonary Disease (COPD) patients between FEV1/FVC and (D) CCSP + BMCs (n = 34), (E) CCSP + PBMCs (n = 40), or (F) CD45 + Collagen-1+ fibrocytes (n = 22). No correlation in Pulmonary Fibrosis (PF) patients between the measured percentage of predicted of FVC and (G) CCSP + BMCs (n = 41), (H) CCSP + PBMCs (n = 53), or (I) CD45 + Collagen-1+ fibrocytes (n = 26). Spearman rank test.
In order to further investigate the biology of these cell populations in end-stage lung disease patients, we next analyzed the potential role of receptor-mediated cytokine-induced migration of CCSP cells.
In order to explore possible mechanisms of recruitment of these cells chemokine receptor expression was investigated for several chemokines implicated in the literature. Chemokine receptor expression by CCSP epithelial-like progenitor cells was first examined by flow cytometry. Sub-populations of CCSP BMC and PBMCs were identified that co-express CCR2, CCR4, CXCR3, or CXCR4 (Figure 5A-D).
(Enlarge Image)
Figure 5.
Chemokine receptor expression by Clara Cell Secretory Protein (CCSP) Cells. Dual expression of chemokine receptors and Clara Cell Secretory Protein (CCSP) on bone marrow cells (BMCs) and peripheral blood mononuclear cells (PBMCs). Representative flow plots are based on PBMCs. (A) CCR2 (B) CCR4 (C) CXCR3 (D) CXCR4. Bars graphs display the mean and standard error.
To further investigate the ability of CCSP cells to migrate in response to chemotactic mediators, in vitro transwell assays were utilized. Migration of bone marrow or peripheral blood cells (BMCs) freshly isolated from end-stage lung disease patients was investigated in response to the chemotactic stimuli RANTES, IP-10, SDF-1, or SCGF-β and compared to untreated cells (Figure 6). A significant migratory response of CCSP cells toward SDF-1 was identified for CCSP PBMCs from control and lung recipient samples, as well as from BMCs from lung recipients, compared to untreated cells in the absence of any chemotactic stimuli. In addition, significant migration in response to SCGF-β was also found for CCSP BMCs and PBMCs isolated from end-stage lung disease patients (p < 0.05), while no significant migratory response was found for CCSP PBMCs isolated from healthy controls (Figure 6).
(Enlarge Image)
Figure 6.
In vitromigration assay. Migration of freshly isolated peripheral blood mononuclear cells (PBMCs) or bone marrow cells (BMCs) in response to chemotactic stimuli Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) (n = 9 control, 11 recipient PBMC, 7 BMC), Interferon gamma-induced protein 10 (IP-10) (n = 9 control, 11 recipient PBMC, 7 BMC), Stromal Derived Factor-1 (SDF-1) (n = 9 control, 13 recipient PBMC, 9 BMC), or Stem Cell Growth Factor-beta (SCGF-β) (n = 4 control, 4 recipient PBMC, 4 BMC), compared to untreated cells. Migrated cells were analyzed for CCSP expression and normalized to total CCSP cells in the starting sample. Kruskal-Wallis test with Dunn's multiple comparison post-hoc analysis, with significance tested against untreated samples (* = p < 0.05, ** = p < .01). Boxes show the median, 25th and 75th percentiles. Whiskers represent the 2.5 and 97.5 percentiles.
To search for other potentially important cell recruitment mediators, a multiplex array was performed on a subset of end-stage lung disease patients' plasma. A total of 17 targets were selected based on biological action and quantified simultaneously (see methods and Additional file 1: Table S3). These results were then analyzed in relation to progenitor cell numbers.
When the plasma protein concentrations were compared across the 3 main end-stage lung diseases, different patterns of expression were noted for some key inflammatory cytokines. Specifically, it was found that IP-10 and MCP-1 are increased in IPF patients, while MIG is increased in across all 3 end-stage groups, and MIF is specifically increased in CF patients when each were compared to lung donor and healthy volunteer control plasma (Figure 7A-D).
(Enlarge Image)
Figure 7.
Plasma cytokine concentrations in end-stage lung disease patients. Comparison of plasma cytokine concentrations between cystic fibrosis (CF) (n = 19), chronic obstructive pulmonary disease (COPD) (n = 16), and pulmonary fibrosis patients (PF) (n = 17) compared to lung donor and healthy volunteer controls (n = 18). Statistically significant differences were found for (A) Interferon gamma-induced protein 10 (IP-10), (B) Monocyte Chemotactic Protein-1 (MCP-1) (C) Monokine-Induced by Gamma Interferon (MIG), and (D) Macrophage Migration Inhibitory Factor (MIF) levels. Kruskal-Wallis test with Dunn's multiple comparison post-hoc analysis. Boxes show the median, 25th and 75th percentiles. Whiskers represent the 2.5 and 97.5 percentiles.
To further investigate the function of plasma protein mediators in progenitor cell recruitment, the relationship between protein concentration and cell numbers was analyzed. The number of CCSP cells in the bone marrow and peripheral blood significantly correlated with the plasma concentration of Stem Cell Growth Factor-beta SCGF-β (Figure 8A) in a range of samples, including lung transplant recipients, donors, and control samples. In addition, it was further found that fibrocyte numbers correlated with the plasma concentration of MCP-1 (Figure 8B).
(Enlarge Image)
Figure 8.
Plasma protein concentration and progenitor cell numbers. (A) The relationship between plasma Stem Cell Growth Factor (SCGF)-β levels and the percentage of CCSP bone marrow cells (BMCs) (n = 44) or CCSP peripheral blood mononuclear cells (PBMCs) in lung transplant recipients (R) and donors (D), and healthy controls (BMC: n = 35R, 9D. PBMC: (n = 49R, 9D, 8H). (B) The relationship between plasma Monocyte Chemotactic Protein (MCP)-1 levels and the percentage of CD45Collagen-1 fibrocytes in lung transplant recipients and donors, and healthy controls (n = 29R, 6D, 2H). Spearman rank test with correlation coefficient.
Results
Identification of Progenitor Populations in End-stage Lung Disease Patients
Circulating bone marrow-derived cell populations were defined by (1) Clara Cell Secretory Protein (CCSP) expression for epithelial-like progenitors and by (2) dual expression of CD45 and intracellular collagen-1 expression for fibrocytes. To validate the expression of CCSP mRNA in the bone marrow and peripheral blood, Taqman PCR was utilized. CCSP mRNA was detected in human bone marrow cells (BMCs) and peripheral blood mononuclear cells (PBMCs) from 3 randomly selected lung transplant recipients, as well as in control lung bronchus tissue, but absent in experimental controls (no reverse transcription and no template controls) (Figure 1A-B). As a further proof-of-principle, peripheral blood cells from a healthy volunteer were isolated and sorted for CCSP by flow cytometery, representing less than 1% of total PBMCs, and then analyzed by PCR. CCSP mRNA was also detected in the pre-sorted population but not in CCSP-negative sorted cells (Figure 1C). The resulting amplification product was further analyzed by gel electrophoresis to confirm the correct amplicon size (Figure 1D), and compared to positive human bronchus tissue mRNA and a mixture of sample mRNAs not subjected to reverse transcription (NRT) to control for genomic DNA contamination. All subsequent quantification of progenitor cells populations was determined by flow cytometry. A representative plot identifying the positively gated populations based on initial isotype staining of 1% is shown for both CCSP cells and CD45Collagen-1 fibrocytes (Figure 1E-F). Further details of the gating strategy have been previously published.
(Enlarge Image)
Figure 1.
Identification of progenitor populations. Taqman PCR measurement of (A) Clara Cell Secretory Protein (CCSP) gene expression in human bone marrow cells (BMCs), (B) peripheral blood mononuclear cells (PBMCs), and (C) PBMCs pre-sorted for CCSP by flow cytometry. No template and no reverse transcription negative controls are included. (D) Gel electrophoresis of PCR product after Taqman-based amplification. Positive control (bronchus) and negative no reverse transcription (NRT) controls are included. (E) Flow cytometry gating for measurement of CCSP PBMCs based on isotype control staining. (F) Flow cytometry gating for measurement of CD45Collagen-1 peripheral blood leukocytes (PBLs) based on isotype control staining.
Bone marrow and peripheral blood samples were collected from patients at the time of lung transplant (n = 154). A summary of relevant lung recipient demographics, including age, gender, diagnosis, diabetes status, BMI, and graft number is presented in Table 1. In addition, blood and bone marrow samples were obtained from lung donors prior to organ recovery (n = 36), the details of which are summarised in Table 2. For logistical reasons, not every patient undergoing lung transplantation or donor procurement could be analyzed in this study. A comparison of recipient and donor demographics from patients included in this analysis compared to the demographics of all those transplanted at our centre in the same time period identified no significant differences in these parameters (Additional file 2: Table S1 and Additional file 3: Table S2).
No significant relationships were identified when these progenitor cell populations were analysed by age, gender, or BMI (data not shown). There were significant differences in the proportion of bone marrow-derived cells populations based on underlying lung disease. An increase in the proportion of CCSP cells was found in the bone marrow of CF patients when compared to lung donors (CF median = 1.33%, Donor median = 0.98, p < 0.05) (Figure 2A). We chose to use lung transplant donors as a comparison group as we had access to sternal marrow biopsy material obtained using identical collection techniques, not easily obtained in any other way. Different cell proportions were also found for CCSP cells in the peripheral blood. Specifically, CF patients had a greater proportion of CCSP cells when compared to lung donors (2.28% CF vs. 1.90% Donor, p < 0.05) (Figure 1B), while BO patients had a significantly lower proportion of CCSP PBMCs compared to donors (0.55% vs. 1.90% Donor, p < 0.05). When circulating CD45collagen-1 fibrocytes were compared between disease groups, an increased proportion was found in both BO patients (p < 0.001) and PF (p < 0.05) patients, when compared to Donors (Median BO = 7.02%, Median PF = 2.07%, Median Donors = 0.85%) (Figure 2C). As the changes in cell numbers appeared to reciprocal, the ratio of fibrocytes-to-CCSP PBMCs was calculated, and a similar pattern was found, where a predominantly fibrotic phenotype is represented in BO and PF, but not in other lung pathologies (Figure 2D).
(Enlarge Image)
Figure 2.
Differential progenitor cell profiles in end-stage lung disease patients. (A) Percentage of bone marrow cells (BMCs) positive for CCSP in each disease group (n = 26 donor, n = 5 Bronchiolitis Obliterans (BO), n = 27 Cystic Fibrosis (CF), n = 34 Chronic Obstructive Pulmonary Disease (COPD), n = 41 Pulmonary Fibrosis (PF), n = 11 Pulmonary Hypertension (PH)). (B) Percentage of peripheral blood mononuclear cells (PBMCs) positive for CCSP in each disease group. (n = 29 donor, n = 6 BO, n = 33 CF, n = 41 COPD, n = 53 PF, n = 13 PH). (C) Percentage of peripheral blood leukocytes (PBLs) positive for CD45 and collagen-1 in each disease group (n = 17 donor, n = 6 BO, n = 14 CF, n = 22 COPD, n = 26 PF, n = 8 PH). (D) Ratio of CCSP PBMCs to CD45collagen-1 fibrocytes in each disease group, compared to lung donors (n = 13 donor, n = 6 BO, n = 10 CF, n = 17 COPD, n = 18 PF, n = 8 PH). Kruskal-Wallis test with Dunn's multiple comparison post-hoc analysis. Boxes show the median, 25th and 75th percentiles. Whiskers represent the 2.5 and 97.5 percentiles.
In order to determine if the proportion of CCSP cells was reflective of the total number of CCSP cells, the absolute cell numbers were determined using total leukocyte counts collected from clinical data (10/L). When absolute cell numbers were compared, the differences in progenitor cell numbers between disease groups were still statistically significant (Additional file 4: Figure S1).
To investigate the relationship between CCSP cells within the bone marrow and the proportion in the peripheral blood, data was analyzed including all disease groups together. A significant correlation was found between the number of bone marrow and peripheral blood CCSP cells (Figure 3A). In contrast, no relationship was found between the number of fibrocytes and either CCSP BMC or PBMCs (Figure 3B-C).
(Enlarge Image)
Figure 3.
Relationship between progenitor cell populations. (A) Correlation between the percentage of Clara Cell Secretory Protein (CCSP) Peripheral Blood Mononuclear Cells (PBMCs) and CCSP Bone Marrow Cells (BMCs) (n = 119 pairs). (B) Lack of correlation between the percentage of CD45Collagen-1 fibrocytes and CCSP BMCs (n = 59 pairs). (C) Lack of correlation between the percentage of CD45Collagen-1 fibrocytes and CCSP PBMC (n = 74 pairs). Spearman rank test with correlation coefficient.
Analysis of clinical disease indicators relative to progenitor cell numbers was done using spirometric lung function values, using the ratio of the forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) (FEV1/FVC) ratio for CF and COPD patients, or based on the percentage of predicted FVC for PF patients. No direct relationships were found between lung function measurements when compared with the total number of epithelial-like progenitors in the bone marrow or peripheral blood or with circulating fibrocytes (Figure 4A-I).
(Enlarge Image)
Figure 4.
Lung function and progenitor cell profiles. No correlation in Cystic Fibrosis (CF) patients between FEV1/FVC and (A) Clara Cell Secretory Protein (CCSP+) bone marrow cells (BMCs) (n = 27), (B) CCSP + peripheral blood mononuclear cells (PBMCs) (n = 32), or (C) CD45 + Collagen-1+ fibrocytes (n = 14). No correlation in Chronic Obstructive Pulmonary Disease (COPD) patients between FEV1/FVC and (D) CCSP + BMCs (n = 34), (E) CCSP + PBMCs (n = 40), or (F) CD45 + Collagen-1+ fibrocytes (n = 22). No correlation in Pulmonary Fibrosis (PF) patients between the measured percentage of predicted of FVC and (G) CCSP + BMCs (n = 41), (H) CCSP + PBMCs (n = 53), or (I) CD45 + Collagen-1+ fibrocytes (n = 26). Spearman rank test.
In order to further investigate the biology of these cell populations in end-stage lung disease patients, we next analyzed the potential role of receptor-mediated cytokine-induced migration of CCSP cells.
Mechanisms of CCSP Progenitor Cell Recruitment
In order to explore possible mechanisms of recruitment of these cells chemokine receptor expression was investigated for several chemokines implicated in the literature. Chemokine receptor expression by CCSP epithelial-like progenitor cells was first examined by flow cytometry. Sub-populations of CCSP BMC and PBMCs were identified that co-express CCR2, CCR4, CXCR3, or CXCR4 (Figure 5A-D).
(Enlarge Image)
Figure 5.
Chemokine receptor expression by Clara Cell Secretory Protein (CCSP) Cells. Dual expression of chemokine receptors and Clara Cell Secretory Protein (CCSP) on bone marrow cells (BMCs) and peripheral blood mononuclear cells (PBMCs). Representative flow plots are based on PBMCs. (A) CCR2 (B) CCR4 (C) CXCR3 (D) CXCR4. Bars graphs display the mean and standard error.
To further investigate the ability of CCSP cells to migrate in response to chemotactic mediators, in vitro transwell assays were utilized. Migration of bone marrow or peripheral blood cells (BMCs) freshly isolated from end-stage lung disease patients was investigated in response to the chemotactic stimuli RANTES, IP-10, SDF-1, or SCGF-β and compared to untreated cells (Figure 6). A significant migratory response of CCSP cells toward SDF-1 was identified for CCSP PBMCs from control and lung recipient samples, as well as from BMCs from lung recipients, compared to untreated cells in the absence of any chemotactic stimuli. In addition, significant migration in response to SCGF-β was also found for CCSP BMCs and PBMCs isolated from end-stage lung disease patients (p < 0.05), while no significant migratory response was found for CCSP PBMCs isolated from healthy controls (Figure 6).
(Enlarge Image)
Figure 6.
In vitromigration assay. Migration of freshly isolated peripheral blood mononuclear cells (PBMCs) or bone marrow cells (BMCs) in response to chemotactic stimuli Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) (n = 9 control, 11 recipient PBMC, 7 BMC), Interferon gamma-induced protein 10 (IP-10) (n = 9 control, 11 recipient PBMC, 7 BMC), Stromal Derived Factor-1 (SDF-1) (n = 9 control, 13 recipient PBMC, 9 BMC), or Stem Cell Growth Factor-beta (SCGF-β) (n = 4 control, 4 recipient PBMC, 4 BMC), compared to untreated cells. Migrated cells were analyzed for CCSP expression and normalized to total CCSP cells in the starting sample. Kruskal-Wallis test with Dunn's multiple comparison post-hoc analysis, with significance tested against untreated samples (* = p < 0.05, ** = p < .01). Boxes show the median, 25th and 75th percentiles. Whiskers represent the 2.5 and 97.5 percentiles.
To search for other potentially important cell recruitment mediators, a multiplex array was performed on a subset of end-stage lung disease patients' plasma. A total of 17 targets were selected based on biological action and quantified simultaneously (see methods and Additional file 1: Table S3). These results were then analyzed in relation to progenitor cell numbers.
When the plasma protein concentrations were compared across the 3 main end-stage lung diseases, different patterns of expression were noted for some key inflammatory cytokines. Specifically, it was found that IP-10 and MCP-1 are increased in IPF patients, while MIG is increased in across all 3 end-stage groups, and MIF is specifically increased in CF patients when each were compared to lung donor and healthy volunteer control plasma (Figure 7A-D).
(Enlarge Image)
Figure 7.
Plasma cytokine concentrations in end-stage lung disease patients. Comparison of plasma cytokine concentrations between cystic fibrosis (CF) (n = 19), chronic obstructive pulmonary disease (COPD) (n = 16), and pulmonary fibrosis patients (PF) (n = 17) compared to lung donor and healthy volunteer controls (n = 18). Statistically significant differences were found for (A) Interferon gamma-induced protein 10 (IP-10), (B) Monocyte Chemotactic Protein-1 (MCP-1) (C) Monokine-Induced by Gamma Interferon (MIG), and (D) Macrophage Migration Inhibitory Factor (MIF) levels. Kruskal-Wallis test with Dunn's multiple comparison post-hoc analysis. Boxes show the median, 25th and 75th percentiles. Whiskers represent the 2.5 and 97.5 percentiles.
To further investigate the function of plasma protein mediators in progenitor cell recruitment, the relationship between protein concentration and cell numbers was analyzed. The number of CCSP cells in the bone marrow and peripheral blood significantly correlated with the plasma concentration of Stem Cell Growth Factor-beta SCGF-β (Figure 8A) in a range of samples, including lung transplant recipients, donors, and control samples. In addition, it was further found that fibrocyte numbers correlated with the plasma concentration of MCP-1 (Figure 8B).
(Enlarge Image)
Figure 8.
Plasma protein concentration and progenitor cell numbers. (A) The relationship between plasma Stem Cell Growth Factor (SCGF)-β levels and the percentage of CCSP bone marrow cells (BMCs) (n = 44) or CCSP peripheral blood mononuclear cells (PBMCs) in lung transplant recipients (R) and donors (D), and healthy controls (BMC: n = 35R, 9D. PBMC: (n = 49R, 9D, 8H). (B) The relationship between plasma Monocyte Chemotactic Protein (MCP)-1 levels and the percentage of CD45Collagen-1 fibrocytes in lung transplant recipients and donors, and healthy controls (n = 29R, 6D, 2H). Spearman rank test with correlation coefficient.