Research paperCytokine profiling of pancreatic fluid using the ePFT collection method in tandem with a multiplexed microarray assay
Research highlights
► We have identified cytokines in pancreatic fluid using ePFT collection in tandem with cytokine microarray technology. ► Cytokines were differentially abundant in the pancreatic fluid of chronic pancreatitis and chronic abdominal paincohorts. ► We observed decreases in concentrations of EGF, IP-10, eotaxin, IL-3, MIP-1a, IL-15, PDGF-AB/BB, and IL-1a in CP specimens.
Introduction
Immunomodulating proteins, such as cytokines, are involved in pancreatic stellate cell activation and pathogenesis of chronic pancreatitis. Cytokines are regulatory proteins that act as intercellular signaling molecules and are produced by various cell types. These low-molecular weight proteins interact with specific cell-surface receptors upon which the ensuing cascades modulate inflammatory and immune responses. Cytokines are generally released in picomolar amounts; however, their concentration can increase over 1000-fold under physiological stress, such as trauma or infection (Cannon, 2000). Cytokines are critical to the development and function of the immune response cascade, as they are often secreted by cells that have undergone cellular stress. This class of proteins includes interleukins (IL), cell signal molecules, such as growth factors (GF), tumor necrosis factor (TNF), and interferons (INF), which trigger inflammation and respond to infections.
In addition, chemokines are a superfamily of small cytokines (8–10 kDa) that act as chemoattractants which guide the migration of cells via corresponding chemokine receptors (Fernandez and Lolis, 2002). These proteins are known to attract leukocytes, neutrophils, monocytes, and other effector cells from the blood to sites of infection or tissue damage (Rottman, 1999). Specifically, chemokines interact with G protein-linked transmembrane receptors, which are selectively found on the surfaces of their target cells. As with cytokines, in general, many chemokines are considered pro-inflammatory. Such proteins can be induced during an immune response to transport cells of the immune system to a site of infection, while others are considered homeostatic and have roles in controlling the migration of cells during normal processes of tissue maintenance or development (Fernandez and Lolis, 2002). Chemokines include eotaxin, fractalkine, growth-regulated oncogene (GRO), interferon-inducible protein (IP), monocyte chemoattractant protein (MCP), myeloma cell metalloproteinase (MDC), macrophage inflammatory protein (MIP), and Rantes.
We have developed and described an endoscopic pancreatic function test (ePFT) that collects pancreatic fluid without cannulation of the pancreatic duct and without the risk of procedure-related injury (Conwell et al., 2003a, Conwell et al., 2003b, Wu and Conwell, 2009). As determined from its electrolyte composition and enzyme activity, the ePFT-collected fluid reproduces the classic acinar and duct cell secretory profiles following hormonal stimulation (Conwell et al., 2002, Conwell et al., 2003a, Conwell et al., 2003b, Stevens et al., 2004a, Stevens et al., 2004b). The ePFT is now considered an acceptable alternative method for the assessment of pancreas secretory physiology (Pollack and Grendell, 2006, Forsmark, 2008). Furthermore, pancreatic fluid collected by the ePFT method is fully amenable to proteomic analysis (Paulo et al., 2011). Thus far, a systematic and comprehensive analysis of immunomodulating proteins, such as cytokines, in pancreatic fluid has not been performed. As the ePFT collection method is a valuable alternative tool to acquire pancreatic fluid even from subjects without pancreas-related disease, we aim to establish this technique, coupled with protein microarray analysis, as a viable strategy to investigate the cytokine profile of pancreatic fluid.
A suspension microarray uses capture antibodies that are coupled to color-coded microspheres and allows the simultaneous analysis of numerous cytokines in a single experiment. Such a multiplexed immunoassay has the advantages of specificity, small sample volumes, and cost-effectiveness. For these reasons, the cytokine protein microarray is both sensitive to the low concentration of cytokines and amenable to high-throughput analysis (Fitzgerald et al., 2008). Although in clinical settings such technology is primarily used for the analysis of urine and blood, proximal body fluids, such as pancreatic fluid, may also benefit from such microarray-based approaches.
The primary objectives of our exploratory investigation are as follows:
- 1)
collect pancreatic fluid with the ePFT method after secretin stimulation,
- 2)
process the pancreatic fluid for cytokine-targeted microarray analysis, and
- 3)
compare cytokine profiles in the pancreatic fluid of chronic pancreatitis and of non-pancreatitis/chronic abdominal pain controls.
The methodology established herein may allow targeted investigations of the cytokines in the pancreatic fluid secretome and broaden our knowledge of pancreatic immune-response and disease pathogenesis.
Section snippets
Study population
The study design was a cytokine analysis of endoscopically collected pancreatic fluid in the setting of an academic center. This protocol was approved by the Institutional Review Board at Brigham and Women's Hospital and Children's Hospital Boston (IRB # 2007-P-002480/1). The study population (Table 1) included adult patients seen in the Center for Pancreatic Diseases at Brigham and Women's Hospital to where they were referred for pancreatic function testing. All subjects underwent the
Demographics
Table 1 displays the characteristics of the 6 subjects in the study cohort. Pancreatic fluid was safely collected via secretin-stimulated ePFT from three controls (CAP) and three patients with definite chronic pancreatitis (CP) based on the M-ANNHEIM classification. Conversely, the M-ANNHEIM criteria for chronic pancreatitis were not met by any of the three individuals with CAP. The mean peak bicarbonate concentrations of the CP and CAP subjects were 43 (abnormal) and 97 (normal) meq/L,
Discussion
We have identified cytokines in secretin stimulated, ePFT-collected pancreatic fluid using a microsphere-based suspension protein microarray assay. To our knowledge, this is the first published report profiling cytokines in ePFT-collected pancreatic fluid. We have compared the levels of 42 different cytokines between chronic pancreatitis (CP) and chronic abdominal pain (CAP) control patients. Our analysis has revealed differences in the cytokine profile of pancreatic fluid between these two
Conclusion
In conclusion, we have successfully identified cytokines in pancreatic fluid using the ePFT collection method in tandem with cytokine microarray technology. Such a workflow is a valuable tool for deciphering the extracellular signaling cascades, which modulate the inflammatory response of the pancreas and which may lead to insights into pancreatic dysfunction. The application of this approach to the various etiologies and clinical stages of pancreatic dysfunction may lead to major insights into
List of abbreviations
- CP
chronic pancreatitis
- CAP
chronic abdominal pain
- ePFT
endoscopic pancreatic function test
- GRO
growth-regulated oncogene
- IP
interferon-inducible protein
- MCP
monocyte chemoattractant protein
- MDC
myeloma cell metalloproteinase
- M-ANNHEIM
Multiple risk factors, Alcohol, Nicotine, Nutrition, Hereditary factors, Efferent duct factors, Immunological factors, and Miscellaneous and metabolic factors
- MIP
macrophage inflammatory protein
- IL
interleukins
- GF
growth factors
- TNF
tumor necrosis factor
- INF
interferons
Competing interests
The authors declare no competing interests.
Author contributions
JP carried out the experiments. JP and DC conceived the study and drafted the original manuscript. JP, HS, and DC participated in its design and coordination. DC, and LL collected the specimens and assisted in experimental design. All authors helped to draft the manuscript and approved the final manuscript.
Acknowledgments
Funds were provided by the following NIH grants: 1 F32 DK085835-01A1 (JP), 1 R21 DK081703-01A2 (DC) and 5 P30 DK034854-24 (Harvard Digestive Diseases Center; DC). In addition, we would like to thank the Burrill family for their generous support through the Burrill Research Grant. We would also like to thank members of the Steen Laboratory at Children's Hospital Boston and Harvard Medical School, in particular John FK Sauld and Robert Everley for their technical assistance and critical reading
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These authors contributed equally to this work.