HMGB-1 is Released Epithelial Cells: Lung Epithelium and Primary Hepatocytes.
Kliment, CR; Liu, S; Sappington, P; Fink, MP.
Medical Scientist Training Program
Department of Critical Care Medicine
University of Pittsburgh Medical Center
BACKGROUND
High Motility Group Box – 1 (HMGB-1) is a nuclear protein involved in gene transcription and
chromatin stabilization. Additionally, HMGB-1 has been identified as a key late inflammatory
mediator in sepsis. Clinically, sepsis is a fatal condition associated with early release of key
cytokines, such as TNF-alpha and Interleukin (IL)-1, and by the late release of HMGB-1. In
response to tissue injury, cytokines are released producing an acute phase response. In the liver,
cytokines produced by resident macrophages and kupfer cells act upon receptors on hepatocytes.
*_**Secretion of HMGB-1, induced by pro-inflammatory cytokines, plays a role in the
dissolution of intercellular tight junctions, as shown in enterocytes, and multiple organ failure.
Cells known to release HMGB-1 include macrophages, monocytes, dendritic cells, neurons,
endothelial cells, smooth muscle cells and enterocytes. HMGB-1 levels increase in the bronchial
alveolar fluid and plasma of patients with acute lung injury, perpetuating lung edema and
neutrophil accumulation. Airway epithelium is part of the defense mechanism designed to
combat thousands of daily challenges to the lungs. These cells do so by pattern recognition
molecules, such as Toll-like receptors (TLR’s) and subsequent cytokine release. Thus, we
hypothesized that airway epithelial cells contribute to HMGB-1 present in the lung after cytokine
challenge and that the autocrine nature of HMGB-1 results in dysfunction of epithelial tight
junctions.
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High Motility Group Box – 1 (HMGB-1) is a nuclear protein involved in gene
transcription and chromatin stabilization. Additionally, it has been identified as a key
late inflammatory cytokine in sepsis (2, 13, 14, 18).
Secretion of HMGB-1, induced by pro-inflammatory cytokines, plays a role in the
dissolution of intercellular tight junctions, as shown in enterocytes (15), and multiple
organ failure.
Cells known to release HMGB-1 include macrophages, monocytes, dendritic cells,
neurons, endothelial cells, smooth muscle cells and enterocytes (5, 7, 23).
Receptors for HMGB-1 include receptors for advanced glycation end-products (RAGE)
and TLR-2 (7, 12).
HMGB-1 levels increase in the bronchial alveolar fluid and plasma of patients with acute
lung injury, perpetuating lung edema and neutrophil accumulation. Airway epithelium is
part of the defense mechanism designed to combat thousands of daily challenges to the
lungs (3).
HMGB-1 increases in hepatic tissue challenged mice and in the serum of sepsis patients
(18). Ethyl pyruvate (EP) has a protective effect against organ injury in various models
inflammatory models (21, 22), protects against liver ischemia-reperfusion injury by
decreasing necrosis and apoptosis of cells () as well as, decreases barrier dysfunction in
endotoxemic mice and cytokine stimulated enterocytes (16).
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We hypothesized that various epithelial cells contribute to HMGB-1 produced after a
challenge or injury to tissue. We looked specifically at airway epithelial cells,
hypothesizing that they contribute to HMGB-1 present in the lung and that the autocrine
nature of HMGB-1 results in dysfunction of epithelial tight junctions.
We also hypothesized that hepatocytes, specialized epithelial cells of the liver, release
HMGB-1 in response to stimulation. Because EP has been shown to be protective
against liver injury, we belief that EP may inhibit the release of HMGB-1 by hepatocytes.
METHODS
Epithelial Cell Culture
A549, alveolar type II lung epithelial cell line, and Calu-3, type I lung epithelial cell line,
(ATCC) were cultured on 24-well collagen coated plates in Hams F12 supplemented media with
10% FBS and Penicillin/Streptomycin. Cells were stimulated with cytomix (10 ng/ml TNFalpha, 10 ng/ml IL-1B, and 1,000 U/ml IFN-gamma) or individual pro-inflammatory cytokines.
Cell supernatants were collected after time course stimulation and HMGB-1 release was
determined by direct sandwich ELISA. Cell viability was assessed using 0.1% trypan blue.
HMGB-1 ELISA
96-well Nunc Maxisorp ELISA Immunoplates were coated with human monoclonal anti-HMG
antibody (R&D Systems) at 1 g/ml in carbonate buffer, overnight at 4C, 50 l/well. Protocol
for Carbonate Buffer, pH 9.6. Make the following stocks: A: 1M NaHCO3 (sodium bicarbonate)
in ddH20, B: 1M NaC03 (sodium carbonate) in ddH20. To make 1L of carbonate buffer add
45.3ml of A and 18.2ml of B, bring up to 1L in ddH20. Sterile filter through 0.2um CA
(cellulose acetate). The pH should be close or equal to 9.6. The plate was blocked with blocking
buffer (PBS + 4% BSA) for 1 hr at 4C (200 l/well). HMGB-1 standards and samples (50
l/well) were incubated for 2 hrs at room temperature (RT). Recombinant full-length HMGB-1
was serially diluted from 1000 ng/ml - 0 ng/ml in blocking buffer. Wells were washed once with
PBS, 200 l/well. Primary antibody detection was with rabbit polyclonal anti-HMGB (BD
Sciences) at 1 g/ml, incubated for 1.5 hrs at 37 C (50 l/well). The plate was washed three
times with fresh PBS + 0.2% Tween-20 for 10 minutes each, 200 l/well then washed once with
PBS for 1 min, 200 l/well. Secondary Ab detection was with Fab’2 donkey anti-rabbit-HRP
(Jackson Immunoresearch) at 1.5 – 3 ug/ml in blocking buffer (50 l/well), incubated for 1 hr at
RT. The plate was washed three times with fresh PBS + 0.2% Tween-20 for 10 minutes each,
200 l/well then washed once with PBS for 1 min, 200 l/well. TMB peroxidase substrate
(Pierce), mixed at equal parts, was used for color development for 15-30 minutes (blue), 100
l/well. The color reaction was terminated with 0.18 M Sulfuric acid (100 ul/well). Absorbance
was read at 450 nm on a spectrophotometric plate reader.
Epithelial Barrier Permeability Assay
Calu-3 cells (Type I epithelia) were used to evaluate HMGB-1 release and tight junction function
by monolayer permeability assays. Briefly, Calu-3 cells were cultured on transwell inserts to
form complete monolayers. DMEM supplemented with Fluorescein isothiocyanate-labeled
dextran (FD4) was sterile filtered and added to the apical compartment of the insert (200 l).
Medium with or without HMGB-1 or cytomix was added to the basal compartment (500l). Cell
supernatants were harvested after 24 hours. Spectrofluorometric analysis was completed on
media from the basal compartment.
Primary Hepatocyte Culture
Primary rat hepatocytes were retrieved by in vivo hepatic collagenase perfusion. Retrieved cells
were washed three times with Hank’s Balanced Salt Solution with Ca2+, centrifuged each time
at 500 g for 5 minutes. Hepatocytes were further purified on a percoll centrifugation gradient.
Cells were seeded onto 24 well collagen-coated plates with round glass slides for imaging, at a
density of 250,000 cells/ml in William’s E medium with 10% FBS and Penicillin/Streptomycin.
After incubation for 18-24 hours, cells were used for experiments.
Cell Stimulation
Cultured hepatocytes were washed once with serum-free William’s E media and fresh serum-free
media was replaced. Cells received media alone, cytomix, Ethyl pyruvate (10 nM) or ethyl
pyruvate plus cytomix for 6, 12, 24 or 36 hours. Cells receiving Ethyl pyruvate with and without
cytomix were pre-treated with Ethyl pyruvate for 2 hrs, which remained on the cells for the
subsequent incubation period. Cytomix was added for the corresponding time span.
Supernatants were collected and centrifuged at 800 g for 5 minutes, 4C. HMGB-1 was
determined by ELISA.
Exosome Collection and Immunohisto-Staining for HMGB-1
Hepatocytes were cultured on collagen-coated slides, as described above. Supernatants were
collected and centrifuged at 800 g for 5 minutes to retrieve exosomes. Cells were fixed for 1
hour with 2% paraformaldehyde at 4C, washed once with cool PBS and stored in PBS for
fluorescent staining. Exosome analysis, immunohistochemisty and cell imaging was completed
by Donna Beers-Stolz, University of Pittsburgh Cell Biology and Physiology Department.
Immunofluorescence confocal microscopy
Fixed cells on slides were washed three times in PBS, then tree times (5 minutes each) with PBS
supplemented with 0.5% bovine serum albumin (PBB). Cells were permeabilized in 0.1%
Triton-X-100 in PBB for 30 minutes, then washed once with PBS containing 0.5% bovine serum
albumin and 0.15% glycine (PBG). Cells were blocked with 2% bovine serum in PBS for 40
min then washed once with PBB. Rabbit anti-HMGB1 antibodies (1:2,000) were added to cells
in PBB incubated at room temperature for 1 hr. Cells were washed four times in PBB then
secondary antibodies (goat anti-rabbit Alexa 488, 1:500 dilution; Molecular Probes, Eugene,
OR), rhodamine-phalloidin (1:250 dilution; Molecular Probes) and the nuclear dye, Draq5
(1:1000 dilution; Biostatus, Leicestershire, UK) were added for 1 hr at room temperature. Cells
were washed three times in PBB then three times in PBS. Cells were mounted using gelvatol (23
g polyvinyl alcohol 2000, 50 ml glycerol, 0.1% sodium azide to 100 ml PBS). Cells were then
viewed on a confocal scanning fluorescence microscope (Olympus Fluoview 1000, Malvern,
NY).
Results/Conclusion
Lung epithelium releases HMGB-1 after stimulation with pro-inflammatory cytokines,
specifically TNF-alpha, IL-1-beta, and IFN-gamma. Our findings suggest that epithelial cells
participate in the production of HMGB-1 in the lung milieu, further contributing to barrier
dysfunction of the lung in sepsis and septic shock.
The liver receives all of the blood as it flows through the body. Therefore, it seems logical that
the specialized cells of this organ respond to injury by producing many inflammatory cytokines,
including HMGB-1 as we have presented.
Future investigation includes further evaluation of the expression of HMGB-1 in other epithelial
cells such as kidney. Additional hepatocyte experiments need to be conducted to confirm the
secretion of HMGB-1 in exosomes and possible re-uptake processes by hepatocytes.
After hepatic ischemia/reperfusion injury in mice, HMGB-1 tissue concentrations increase in a
biphasic , time-dependent manner (19, 20).
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