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Transforming growth factor beta 1 (TGFβ1) is a major fibrogenic cytokine (1). We have previously shown that, in children with cystic fibrosis liver disease (CFLD), TGFβ1 protein is increased in perifibrotic hepatocytes and correlates strongly with histologic quantitation of hepatic fibrosis (2). Surprisingly, in a small pilot study of children with CFLD, plasma TGFβ1 was decreased compared with values for “well” children with cystic fibrosis (CF; normal serum aminotransferases, forced expiratory volume in 1 second [FEV1] > 80% of expected). The purpose of the present study was threefold: 1) to assess plasma TGFβ1 in children with CFLD from three additional medical centers to investigate the generalizability of our pilot data, 2) to correlate plasma TGFβ1 with platelet factor 4 (PF4) because both are released from the alpha granules of platelets during platelet activation, and 3) to assess plasma TGFβ1 in patients with CFLD (FEV1 <40% of expected), because TGFβ1 expression is increased in lung tissue obtained by bronchoalveolar lavage from patients with cystic fibrosis (3).
MATERIALS AND METHODS
Cystic fibrosis patients
Three different groups of patients with CF from four different medical centers (Johns Hopkins, Kaiser Permanente, Columbus Children's, and the University of Colorado) were enrolled in the study after informed consent was obtained.
- Group 1 (CFLD; patients with liver disease): serum alanine aminotransferase more than twice the upper limit of normal for more than 3 months; the presence of hepatomegaly, splenomegaly, or both (4); or both (n = 35; 14 ± 1 years of age).
- Group 2 (patients with CF and no obvious liver disease [CFNLD]): serum alanine aminotransferase less than twice the upper limit of normal, absence of hepatomegaly and splenomegaly, FEV1 > 50% predicted (n = 19; 13 ± 1 years of age).
- Group 3 (CFPD; patients with advanced pulmonary disease): serum alanine aminotransferase less than twice the upper limit of normal, absence of hepatomegaly and splenomegaly, and FEV1 < 40% predicted (n = 8; 26 ± 5 years of age).
The study was approved by institutional review boards at all centers.
Two groups of subjects served as healthy age-group controls, because we had previously shown that both plasma TGFβ1 and plasma PF4 are high in young infants and decrease with advancing age, exhibiting strong inverse correlations with age in years. Groups were healthy adolescents (n = 10; 13 ± 1 years of age) and healthy adults (n = 9; 30 ± 2 years of age) without evidence of either liver or pulmonary disease. These normative data were published previously (2).
Serum alanine aminotransferase, aspartate aminotransferase, total serum bilirubin, and platelets were assessed by standard autoanalyzers. Blood was collected percutaneously and care was taken to obtain plasma as atraumatically as possible to minimize artifactual TGFβ1 derived from endothelium and platelet stores. The first 2 mL blood were discarded and 5 mL were drawn gently into a separate syringe and immediately transferred to a chilled heparinized tube. Vacuum was released from the tube before adding the sample. Samples were centrifuged at 1000 g for 30 minutes, within 1 hour of collection. The plasma supernatant fraction was stored at −70°C until assay. Plasma samples obtained at institutions outside Johns Hopkins were shipped frozen on dry ice to Johns Hopkins for assay of TGFβ1 and PF4. Plasma TGFβ1 was assayed by the R and D Quantikine quantitative sandwich assay 7 (D Systems, Minneapolis, MN) and PF4 by enzyme-linked immunosorbent assay (American Bioproducts, Parsippany, NJ), as we have previously described (2).
Relationships among plasma TGFβ1 and PF4 for the various groups were examined using linear regression. Plasma TGFβ1 was compared between CFLD and CFNLD using the Mann–Whitney rank sum test. A one-way analysis of variance followed by pairwise multiple comparison procedures (Dunn's method) was used to analyze plasma TFGβ1 and PF4 and other laboratory analytes between groups. The alpha level for statistical significance was 0.05. Continuous data results are reported as means ± standard error of mean.
Clinical and laboratory data in the three groups of patients with CF are shown in Table 1. The CFLD and CFNLD patients were of comparable ages, and the CFPD patients were older and had a markedly decreased FEV1. Serum alanine aminotransferase values in the CFLD patients were increased compared with that of the other two groups. Total serum bilirubin (see Table 1), alkaline phosphatase, and platelet counts (not shown) were normal and did not differ between CFLD and CFNLD groups. The FEV1 in CFLD patients (81% ± 4%) did not differ from that of CFNLD patients.
Plasma TGFβ1 values in the CFPD patients were markedly increased compared with values for healthy adults of comparable age (P < 0.004;Fig. 1). In contrast, plasma TGFβ1 values did not differ between CFLD, CFNLD, and healthy teens, all of comparable ages. Although the mean value for CFLD was lower than for CFNLD, there was significant overlap and the difference was not statistically significant (P = 0.1025).
Plasma PF4 values in the CFPD patients also were markedly increased compared with values for healthy adults of comparable age (P < 0.0001;Fig. 2). In contrast, plasma PF4 values were lower in CFLD patients compared with values for healthy adolescents (P < 0.001), with no difference between CFLD and CFNLD of comparable age.
Figure 3 demonstrates the strong correlation between TGFβ1 and PF4 for the entire group of data. However, the correlations between TGFβ1 and PF4 within the various groups were not significant: CFLD, r = 0.566, P = 0.069; CFPD, r = 0.561, P = 0.148; CFNLD, r = −0.338, P = 0.219.
Our data for plasma TGFβ1 values for this larger population of children with CFLD were not consistent with our previous findings (2) of significantly decreased values in 11 children with CFLD (2 ± 1 ng/mL) compared with values for 14 children with CF and no obvious liver disease (12 ± 1 ng/mL; P < 0.05). In our previous study (2), plasma TGFβ1 in 11 subjects with CFLD (ages 16 ± 4 years) was depressed versus values for 14 CFNLD patients (aged 12 ± 1 years; 2 ± 1 ng/mL vs. 12 ± 1 ng/mL; P < 0.05). With the present study, with expansion of the study to four different medical centers, 35 patients with CFLD (ages 14 ± 1 years of age) and 19 with CFNLD (13 ± 1 years of age), plasma TGFβ1 values were 7 ± 1 ng/mL versus 12 ± 4 ng/mL, respectively, not a significant difference. This lack of a significant difference in plasma TFGβ1 in the larger group of children with CFLD versus the CFNLD patients could be explained by the somewhat younger ages of the larger CFLD group in comparison with the pilot CFLD group by differences in sample handling from one center to the other, leading to increased contamination of some of the samples by platelet-derived TGFβ1, because the CFLD in the larger group of patients was more heterogeneous than in the smaller group, or both. Regardless of which explanations are true, the value of the larger study is that it is quite clear that plasma TGFβ1 is not a useful marker of CFLD in children.
Because we did not consider it ethical to perform liver biopsies systematically on all of our children with CFLD, we acknowledge that the previously published definition of CFLD (4) we used for this study could encompass pathologically distinct forms of liver disease. Lacking hepatic histologic analysis, we deliberately selected a broad definition of CFLD. However, in our previous pilot study (2), plasma TGFβ1 was depressed in patients with CFLD whether the available liver biopsy specimens demonstrated cirrhosis, only focal fibrosis, or macrovesicular steatosis.
Our data suggest that there is a need for highly sensitive and specific markers of CFLD other than plasma TGFβ1. Depending on the diagnostic criteria used, CFLD occurs in 17% to 42% of patients with CF (6–10) and is considered a major complication of CF that may limit survival and the quality of life of affected patients (11). Cirrhosis often develops before age 10 years in susceptible individuals (9). Noninvasive means of early detection are needed urgently to serve as reliable means of following up response to therapy; but the diagnosis and detection of CFLD has proven to be quite difficult.
Furthermore, in our previous study in children with CFLD and other types of fibrosing liver disease, we showed that plasma and liver TGFβ1 do not correlate, even though the liver biopsies from some CFLD patients demonstrated strong staining for TGFβ1 protein in perfibrotic hepatocytes (2).
Perhaps the data of greatest clinical usefulness in this study are the findings of markedly elevated plasma TGFβ1 and PF4 in the cystic fibrosis patients with advanced pulmonary disease. Although there are no previous reports of these plasma markers of platelet activation in this group of patients, there is both in vitro data and histopathologic data to suggest that there is platelet activation in such patients. Corrin et al. (12) noted abundant expression of both intracellular and extracellular TGFβ1 in the alveoli in the organizing pneumonia in CF. Awad et al. (13) noted that, in patients requiring lung transplantation for a fibrotic lung condition, there was an increase in the frequency of a high-producer TGFβ1 allele. This allele was associated significantly with pretransplant fibrotic pathologic features and was associated with allograft fibrosis in transbronchial biopsies when compared with controls. Plasma TGFβ1 values were not assessed.
There are no reports of PF4 in CF patients with pulmonary disease. However, PF4 is released from platelets during shear-induced activation (14), as may be occurring in CF lungs. A cationic protein, PF4 has been shown to induce airway hyperresponsiveness in a rat model, a phenomenon that is dependent on the positive charge of the protein, because low molecular weight heparin neutralized both the airway hyperresponsiveness and the positive charge (15). Interestingly, symptomatic patients with bronchial asthma do not exhibit increased plasma PF4, suggesting that PF4 is more a marker of inflammatory, fibrotic lung disease than of airway hyperresponsiveness per se (16). Buyukasik et al. (17) showed that plasma PF4 levels were increased in patients with pulmonary tuberculosis and correlated well with the extent of pulmonary lesions on chest radiography. These authors suggested that platelets may play a pathophysiologic role in this pulmonary process. Our observations of high plasma TGFβ1 and PF4 values in healthy infants and adolescents compared with values for healthy adults (2) highlight the importance of careful selection of controls in future studies of the role of platelet factor 4 in CFPD.
We conclude that, although depression of plasma TGFβ1 is not a useful marker in the detection of CFLD, plasma TGFβ1 and PF4 are elevated in patients with advanced lung disease secondary to cystic fibrosis. These data suggest that platelet activation may either play a role in or be a marker of pulmonary disease in patients with CF, or both. (5)
The authors than Beryl Rosenstein, MD, for patient referral; Margaret Wilson for expert secretarial assistance; P. Yvonne Barnes for assistance with graphics; and Lalage Wakefield, PhD, for advice regarding the TGFβ1 and PF4 assays.
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