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. 2012 Feb;61(2):436-46.
doi: 10.2337/db11-0853. Epub 2011 Dec 30.

Expression and regulation of chemokines in murine and human type 1 diabetes

Suparna A Sarkar  1 , Catherine E Lee, Francisco Victorino, Tom T Nguyen, Jay A Walters, Adam Burrack, Jens Eberlein, Steven K Hildemann, Dirk Homann
Affiliations

Expression and regulation of chemokines in murine and human type 1 diabetes

Suparna A Sarkar et al. Diabetes. 2012 Feb.

Abstract

More than one-half of the ~50 human chemokines have been associated with or implicated in the pathogenesis of type 1 diabetes, yet their actual expression patterns in the islet environment of type 1 diabetic patients remain, at present, poorly defined. Here, we have integrated a human islet culture system, murine models of virus-induced and spontaneous type 1 diabetes, and the histopathological examination of pancreata from diabetic organ donors with the goal of providing a foundation for the informed selection of potential therapeutic targets within the chemokine/receptor family. Chemokine (C-C motif) ligand (CCL) 5 (CCL5), CCL8, CCL22, chemokine (C-X-C motif) ligand (CXCL) 9 (CXCL9), CXCL10, and chemokine (C-X3-C motif) ligand (CX3CL) 1 (CX3CL1) were the major chemokines transcribed (in an inducible nitric oxide synthase-dependent but not nuclear factor-κB-dependent fashion) and translated by human islet cells in response to in vitro inflammatory stimuli. CXCL10 was identified as the dominant chemokine expressed in vivo in the islet environment of prediabetic animals and type 1 diabetic patients, whereas CCL5, CCL8, CXCL9, and CX3CL1 proteins were present at lower levels in the islets of both species. Of importance, additional expression of the same chemokines in human acinar tissues emphasizes an underappreciated involvement of the exocrine pancreas in the natural course of type 1 diabetes that will require consideration for additional type 1 diabetes pathogenesis and immune intervention studies.

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Figures

FIG. 1.
FIG. 1.
Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (microarray analysis). Purified human islets obtained from healthy organ donors were cultured for 24 h in the absence (Control) or presence of individual recombinant human cytokines IL-1β, TNFα, or IFNγ or combinations thereof (MIX) prior to microarray analysis as described in RESEARCH DESIGN AND METHODS. The normalized intensity (log scale) from data obtained on the HG U133 Plus 2.0 Affymetrix chip is shown. Each data point is the mean ± SE of three to four observations. Cytokine cocktail (MIX)-induced expression by a factor of >30 was observed for CCL5, CCL8, CCL22, CX3CL1, CXCL9, and CXCL10 (asterisks indicate significant differences between control and cytokine-treated islets).
FIG. 2.
FIG. 2.
Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (qRT-PCR analysis). Chemokine transcript expression in human islets cultured as described in the legend to Fig.1 and RESEARCH DESIGN AND METHODS was measured by qRT-PCR using a 5′-nuclease assay and FAM dye–labeled TaqMan MGB probes with two PCR primers. Endogenous HPRT1 was used for normalization. Data (mean ± SE; four donors) was quantified using the 2–ΔΔ CT method and expressed relative to an islet sample incubated in medium alone. For direct comparison, a value of 1.0 (dotted line) was assigned to TNFα-induced (CCL5, CXCL9/10, and CX3CL1) or IL-1β–induced (CCL22) chemokine transcripts. Asterisks indicate significant differences between control and cytokine-treated islets.
FIG. 3.
FIG. 3.
Immunofluorescent localization of chemokines in cultured human islets. Islets were cultured for 24 h in MIX, fixed with 4% paraformaldehyde (PFA), embedded in paraffin, sectioned, and stained by the immunofluorescent procedure. Insulin (Cy2), glucagon (AMCA), and chemokine (Cy3) immunofluorescent reactivity are shown. Please note that a certain loss of insulin-staining intensity typically occurs as a result of the nature of our islet isolation and culture procedure; however, the integrity of β-cell function was verified in vivo as detailed in RESEARCH DESIGN AND METHODS. The figure is representative of two experiments performed with two different donor islets. INS/GCG, insulin/glucagon; MERGE, merged images. Scale bar: 15 μm. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Chemokine expression in the RIP-GP model of virus-induced type 1 diabetes. RIP-GP mice were infected with LCMV, and their pancreata were harvested 7 days later and processed for immunohistological analysis as detailed in RESEARCH DESIGN AND METHODS. Note the minimal or absent expression of CCL22 and CXCL9, the preferential expression of CCL8 and CXCL10 by β-cells, as well as CX3CL1 production by α-cells; the right-hand column features magnified sections of merged CCL8, CXCL10, and CX3CL1 stains. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 5.
FIG. 5.
Chemokine expression in the NOD model of spontaneous type 1 diabetes. A: Chemokine mRNA transcript expression was quantified in islets isolated from Balb/c (n = 6) as well as 8- and 13-week-old female NOD mice (n = 6) by qRT-PCR. Data were quantified using the 2–Δ Δ CT method expressed as means ± SD (n = 3) and normalized to housekeeping Hprt and Balb/c samples (calibrator). Relative chemokine mRNA expression is displayed in relation to Balb/c samples (dotted line); asterisks indicate significant differences between control and cytokine-treated islets. B: CXCL10 production by pancreatic β-cells as a function of female NOD age was determined as detailed in RESEARCH DESIGN AND METHODS. Note the weak CXCL10 staining in acinar tissues in 4-week-old NOD mice, preferential colocalization with insulin in 12-week-old NOD mice, and complete absence of CXCL10 at 23 weeks of age. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 6.
FIG. 6.
Chemokine expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Pancreatic sections from healthy control subjects (case identification nos. 6117, 6112, and 6115) and type 1 diabetic donors (case identification nos. 6052 and 6087) were acquired through the nPOD program and stained for insulin, glucagon, and chemokines as detailed in RESEARCH DESIGN AND METHODS. Note the presence of some CCL5, CCL8, and CXCL9 in diabetic donors but their absence in healthy control samples. Only very faint CX3CL1 staining was observed in one of the type 1 diabetic samples (identification no. 6087). Scale bar: 20 μm. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 7.
FIG. 7.
In situ CXCL10 expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Combined insulin, glucagon, CD45, and CXCL10 stains were performed as detailed in RESEARCH DESIGN AND METHODS. Note the absence of CXCL10 staining in the healthy control subjects (case identification no. 6112) but ready presence in type 1 diabetic samples (identification nos. 6087 and 6052), sometimes in close association with infiltrating leukocytes (CD45+). To confirm the pattern of exocrine CXCL10 staining, we included a sample from an additional diabetic donor with clinically confirmed pancreatitis (identification no. 6036). (A high-quality digital representation of this figure is available in the online issue.)
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