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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-03-0918.
IMMUNOBIOLOGY
From the Department of Trauma Surgery, University
Hospital Mannheim, Faculty of Clinical Medicine Mannheim of the
Ruprecht-Karls University Heidelberg, Mannheim, Germany;
and Department of Anaesthesiology, University Hospital Mannheim,
Faculty of Clinical Medicine Mannheim of the Ruprecht-Karls University
Heidelberg, Mannheim, Germany.
Ubiquitin is suggested to play a key role in essential
intracellular functions, such as heat shock response, protein
breakdown, and regulation of immune responses. Ubiquitin has also been
detected in the extracellular space, but the function and biologic
significance is unclear. We describe a new function of extracellular
ubiquitin and show that extracellular ubiquitin specifically inhibits
ex vivo secretion of tumor necrosis factor- Ubiquitin, a small (8.6-kDa), heat-stable, and
highly conserved 76-amino acid protein in all eukaryotic cells has
originally been discovered as an immunopoietic polypeptide from
thymocytes.1,2 Further research has suggested that
ubiquitin plays a key role in essential intracellular functions such as
cell differentiation, cell cycle control, heat shock response, and
regulation of immune responses.3-5 Traditionally, the most
important function of ubiquitin is believed to be regulation of protein
turnover by the ubiquitin-proteasome pathway.3-6
Furthermore, ubiquitin has also been detected in the extracellular
space. Compared with the normal range, significantly increased
ubiquitin levels have been described in serum or plasma during
parasitic infections,7 in alcoholic liver
cirrhosis,8 in type 2 diabetes,9 in hairy
cell leukemia,10 and in patients with renal failure and
hemodialysis treatment,11,12 but data on the potential
biologic functions of extracellular ubiquitin are rare. Besides
possible lymphocyte-differentiating properties, extracellular ubiquitin
has been reported to inhibit platelet activities13 and IgG
production in splenocyte cultures,14 to synergistically
effect lipopolysaccharide (endotoxin; LPS)-induced tumor necrosis
factor- In the present study, we describe a new biologic function of
extracellular ubiquitin and show that exogenous ubiquitin specifically inhibits the TNF- Healthy blood donors and critically ill patients
Sepsis patients (8 women, 16 men) fulfilled the Criteria of the
American College of Chest Physicians/Society of Critical Care Medicine
consensus conference20 (10 patients for sepsis, 7 patients for severe sepsis, and 7 patients for septic shock). The age of the
sepsis patients was 52 ± 18 years (mean ± SD). The source of
infection was pneumonia in 17 patients, peritonitis in 6 patients, and
pancreatitis in 1 patient. Five patients with septic shock died. All
patients requiring surgical intervention received standard surgical
care and postoperative intensive care unit treatment. Blood was
collected in plastic tubes (NH4-heparin (9 mL) and serum (9 mL) tubes, Sarsted, Nümbrecht, Germany) along with the
routine baseline laboratory work-up. Whole blood collected in a serum tube was separated and the sera were aliquoted and stored frozen at
Furthermore, blood from mongrel swine (n = 3, 35-55 kg body
weight; kindly provided by Prof Dr K. G. Proctor, Department of Surgery, Ryder Trauma Center, University of Miami, FL) and mice (n = 3, 25-35 g body weight; kindly provided by Dr A. El-Haddad, Department of Surgery, University of Miami) was collected in an NH4-heparin tube. Whole blood collected in an
NH4-heparin tube was immediately used for culture
experiments and for isolation of PBMNCs.
Isolation of PBMNCs and cell cultures
Proteins and antibodies Ubiquitin was purchased from Sigma (U6253). Prior to use in cell cultures, ubiquitin was boiled for 5 minutes, placed on ice for 5 minutes, and centrifuged. The pellet was removed and the supernatant, which contains the heat-stable ubiquitin, was further used. Human recombinant interleukin-10 (IL-10; I9276) was purchased from Sigma. Rabbit antiubiquitin antiserum (AS; U5379), ubiquitin-fluorescein conjugate (U5504), and goat antiserum to rabbit IgG (R8633) were purchased from Sigma. Monoclonal mouse antiubiquitin antibody (UbP4D1) and goat polyclonal antiubiquitin antibody (UbN19) were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). Peroxidase-linked antirabbit and antimouse IgG was obtained from Amersham-Pharmacia (Freiburg, Germany).Immunoassays Ubiquitin.
Quantification of ubiquitin concentrations in serum and urine
specimens was performed with a competitive-binding immunoassay, in
which ubiquitin-fluorescein conjugate and ubiquitin in the test sample
compete for a limited number of binding sites in the antiubiquitin
antiserum. Two to 4 dilutions of each serum/urine sample were measured
in duplicate. In brief, 100 µL ubiquitin-fluorescein conjugate, 100 µL test sample, and 100 µL rabbit antiubiquitin antiserum were
transferred to plastic tubes, mixed, and incubated for 60 minutes at
room temperature in the dark. After incubation, 1 mL goat antiserum to
rabbit IgG was added to the test tubes, the solution was centrifuged
for 15 minutes at 4°C, and the supernatant was removed. The pellet
was resuspended in 2 mL 0.1 N NaOH and 2% sodium dodecyl sulfate
(SDS), and the fluorescence ( TNF- Immunoblotting Following SDS-polyacrylamide gel electrophoresis (SDS-PAGE), serum or urine samples were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Hybond-P; Amersham-Pharmacia). After blocking residual binding sites on the membrane with 5% (wt/vol) nonfat dried milk powder (Milupa, Friedrichsdorf, Germany), 0.1% Tween 20 (Sigma) in PBS immunoblotting was performed with antiubiquitin AS (1:200, vol/vol) and monoclonal UbP4D1 (1:500, vol/vol) using a corresponding second horseradish peroxidase-labeled antibody (1:10 000, vol/vol, and 1:5000, vol/vol), respectively; Amersham Biosource, Freiburg, Germany). Immunoreactive proteins were visualized with a enhanced chemiluminescence (ECL-Plus) detection system (Amersham Pharmacia) using the ImageMaster VDS-CL video system (Amersham Pharmacia).Affinity chromatography Antiubiquitin affinity chromatography was performed using the rabbit antiubiquitin AS (Sigma). HiTrap NHS-activated columns (1-mL column, 7-mm inner diameter × 25-mm column height; Amersham Pharmacia) were incubated with rabbit antiubiquitin AS (4 mg/mL in 0.2 M NaHCO3, 0.5 M NaCl, pH 8.3) for 30 minutes at ambient temperature. After incubation, the column was washed and deactivated with several volumes of 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 (buffer A), 0.1 M acetate, 0.5 M NaCl, pH 4 (buffer B), and again with buffer A with a flow rate of 1 mL/min. Following 25 minutes of incubation in buffer A at ambient temperature, the column was washed again and was then equilibrated with RPMI 1640 (Gibco BRL). Patient serum 1:1 (vol/vol) in RPMI 1640 was applied to the column and was incubated for 30 minutes. The run-through was collected and the column was washed with several volumes of RPMI 1640. The column was eluted with a 5-step pH gradient of each 2-column volumes of 0.2 M glycine at pH 7, pH 6, pH 5, pH 4, and pH 3 with a flow rate of 1 mL/min. Fractions of 1 mL were collected. Immediately after elution, the fractions were neutralized to pH 7.5 and were used in cell culture experiments.mRNA quantification TNF- mRNA levels in endotoxin-stimulated PBMNCs
(106 PBMNC/mL) were quantified using a commercially
available colorimetric microplate assay kit (Qantikine mRNA; R & D
Systems) according to the manufacturer's instructions.
The lower detection limit is 3.2 amol TNF- mRNA/mL.
Other procedures and substances Protein was determined with a protein assay kit (P5656, Sigma) using bovine serum albumin as standard. Protein standards for gel electrophoresis were purchased from Amersham Pharmacia. Zymosan A (Z4250) was purchased from Sigma. Heat-killed Staphylococcus aureus (clinical isolates, autoclaved) was kindly provided by Prof Dr H. Hof (Institute of Medical Microbiology, University Hospital Mannheim, Germany). Cells were counted with a XR-21 automatic multichannel hematology cell counter (Sysmex, Norderstadt, Germany).Statistics If not otherwise mentioned, data are expressed as the mean ± SEM. Spearman correlation coefficient (rs), Student t test, and the Bonferroni corrected one-way analysis of variance (ANOVA) for multiple comparisons were calculated with the SPSS for Windows Release 10.0.7 program (SPSS, Chicago, IL). A 2-tailed P < .05 was considered significant. Standard curves of the assays and dose-related effects of exogenous ubiquitin were analyzed by linear and nonlinear regression analysis using the GraphPad Prism program (version 1.0, 1994, GraphPad Software).
Exogenous ubiquitin inhibits LPS-induced TNF-
secretion of human whole blood and PBMNC cultures stimulated with and without LPS. TNF- was not detectable in LPS free whole blood and
PBMNC cultures incubated with 0 to 1 µg/mL exogenous ubiquitin (not
shown). As estimated with the MTT assay viability was more than 90% in
all PBMNC cultures (not shown). In whole blood and PBMNC cultures with
LPS, exogenous ubiquitin significantly inhibited the TNF- secretion
in a dose-dependent manner (correlation coefficients: whole blood
rs = 0.92; PBMNC rs = 0.96; Figure
1). Maximal inhibition of the TNF-
production was found at a concentration of 500 ng/mL exogenous
ubiquitin in both whole blood and PBMNC cultures. As expected, no
difference in inhibitory activity between boiled and untreated
ubiquitin was detectable (Table 1).
Therefore, boiled ubiquitin was used throughout the study to
heat-inactivate and exclude possible biologically active compounds at
concentrations below the detection limit of SDS-PAGE in the ubiquitin
solution. Kinetics of the LPS-stimulated TNF- production of whole
blood and PBMNCs showed that exogenous ubiquitin did not influence the time course of the TNF- secretion within an incubation period of 0 to 16 hours. To exclude interference of exogenous ubiquitin with the
immunologic detection of TNF- in the cell cultures, ubiquitin was
added to whole blood and PBMNC cultures (n = 8) after 4 hours of LPS
stimulation. Compared with the control measurements without ubiquitin,
in the presence of 500 ng/mL and 1000 ng/mL ubiquitin, the recovery of
TNF- was 97% ± 1.7% (mean ± SEM) and 96% ± 3%
(mean ± SEM), respectively.
To further confirm the inhibitory effect of exogenously added ubiquitin
on LPS-stimulated TNF- In line with the findings in human whole blood, exogenous ubiquitin was
found to inhibit the LPS- induced TNF- In contrast to the LPS-evoked TNF-
Ubiquitin serum and urine concentrations in healthy individuals and critically ill patients Determination of ubiquitin levels in serum, plasma, and whole blood derived from the same blood specimen revealed equal concentrations in each sample (Table 3), indicating no relevant ubiquitin release during blood clotting or sample preparation. This is corroborated by the finding that ubiquitin serum concentrations in a donor's blood specimen were determined to be 84 ng/mL when serum was separated immediately, 90 ng/mL when serum was separated 1 hour after collecting the blood in a serum tube, and 90 ng/mL after 4 hours, respectively.
Ubiquitin was detectable in 27 of the 35 serum samples from healthy
blood donors, in all serum samples from multiply injured patients on
day 0 and 1 after trauma, and in all serum samples from sepsis
patients. In healthy individuals ubiquitin serum concentrations were
determined to be 58 ± 48 ng/mL (mean ± SD). Compared with healthy individuals, the ubiquitin serum concentrations were found to
be 6-fold elevated in multiply injured patients (n = 23) on day 0 after trauma (359 ± 177 ng/mL, mean ± SD) as well as in 24 sepsis patients (327 ± 203 ng/mL, mean ± SD; Figure
3A).
Furthermore, we detected ubiquitin concentrations in urine specimens. Similar to the findings in serum specimens, the urine ubiquitin concentrations were found to be in the same range of magnitude with an urine ubiquitin concentration of 41 ± 22 ng/mL (mean ± SD) in healthy volunteers and a 4.5-fold increased urine ubiquitin concentration (180 ± 166 ng/mL, mean ± SD) in sepsis patients. In addition, we performed immunoblot analysis of patient and healthy donor serum and urine specimens. As determined from control experiments using ubiquitin as a standard, the detection limit was 1 ng ubiquitin using the antiubiquitin AS and 20 ng ubiquitin using the monoclonal UbP4D1 (not shown). Using both antibodies, patterns of detectable ubiquitin immunoreactive proteins were found to be identical (Figure 5B, lanes 1 and 5, lanes 2 and 6, and lanes 3 and 8). As shown in Figure 3, no or only a faint band corresponding to free ubiquitin was detectable in healthy donor samples (Figures 3B lane 1 and 3C lane 2), whereas patient serum and urine samples contained a strong band corresponding to free ubiquitin (Figure 3B-C). Although unspecific binding cannot be excluded for each of the numerous high-molecular-weight bands visualized using both the antiubiquitin AS and the monoclonal UbP4D1, obvious differences between patient and healthy donor serum samples were not detectable except for free endogenous ubiquitin (Figures 3B lanes 1 and 3 and 5B lane 1). Comparison of ubiquitin serum concentrations with the LPS-induced
whole blood TNF- production, we compared ubiquitin serum
concentrations with the whole blood TNF- response to LPS in healthy
individuals and trauma patients. As shown in Figure 4A, high ubiquitin serum concentrations
are significantly associated with low TNF- concentrations in
LPS-stimulated whole blood from healthy donors and severely injured
patients (n = 62, rs =  0.263; P = .018).
In severely injured trauma patients, the ubiquitin serum concentrations
on days 0 to 14 resemble a mirror image of the LPS-induced whole blood
TNF- production.
Antiubiquitin antibodies neutralize the inhibitory activity for
TNF- -producing capacity of volunteers' whole blood and PBMNCs.22-25 To address the
involvement of ubiquitin in this context, we tested the effect of
antiubiquitin antibodies in whole blood and PBMNC cultures incubated
with and without patients' serum (Figure 4). In a first series of cell culture experiments, we examined the potential neutralizing effect of
antiubiquitin antibodies on the inhibitory activity of ubiquitin on the
LPS-induced TNF- secretion. Antiubiquitin AS was found to neutralize
the effect of ubiquitin dose dependently at a dilution of 1:100 and
1:10 without effects on whole blood and PBMNCs cultured in the absence
of exogenous ubiquitin. Moreover, the tested monoclonal (UbP4D1 diluted
1:1000) and polyclonal antiubiquitin antibody (UbN19 diluted 1:1000)
neutralized the inhibitory effect of exogenous ubiquitin on the
LPS-stimulated TNF- release in whole blood and PBMNC cultures. None
of these antibodies affected the TNF- secretion of cell cultures
without exogenous ubiquitin. To further exclude unspecific stimulation
induced by immune complexes, the LPS-induced TNF- secretion of whole
blood and PBMNCs was tested in cocultures with exogenous human
recombinant IL-10 and antiubiquitin antibodies. The antiubiquitin
antibodies did not influence the IL-10-induced inhibition of the
LPS-stimulated TNF- secretion (not shown). In the second series of
experiments, whole blood and PBMNCs from healthy donors were cultured
in the presence of serum from trauma (Figure 4E) and sepsis (Figure 4F)
patients and the effect of the antiubiquitin antibodies was examined.
As expected, serum from trauma patients (mean ubiquitin level,
330 ± 99 [SD] ng/mL) reduced LPS-stimulated TNF- Incubating whole blood and PBMNCs in the presence of serum from sepsis
patients (mean ubiquitin level, 393 ± 179 [SD] ng/mL) inhibited
the LPS-induced TNF- In contrast to PBMNC cultures, the neutralizing effect of
antiubiquitin AS on the inhibition induced by serum from sepsis patients on LPS-induced whole blood TNF- Endogenous ubiquitin regulates the inhibitory activity for TNF- secretion was abolished. In line with the
biologic activity, immunoblot analysis of the run-through showed that
free endogenous ubiquitin was removed from the patients' serum.
Elution of bound proteins from the antiubiquitin column was performed
by acidification. Inhibitory activity for LPS-induced TNF- secretion
was found in the eluted fractions, with a maximal inhibitory effect of
the fractions at pH 4. The inhibitory activity measured in fraction 4 was similar to the effect of patients' serum. Immunoblotting of the
fractions containing the maximal inhibitory activity showed a single
band corresponding to free ubiquitin, whereas the high-molecular-weight bands were detectable in the unadsorbed fractions.
Antiubiquitin antibodies restore reduced TNF- -producing capacities (Figure 6).
In healthy donor blood antiubiquitin antibodies did not influence the
TNF-
In this study we found that exogenous ubiquitin specifically
inhibits the LPS-induced TNF- Based on the findings that exogenous ubiquitin influences neither
LPS-evoked IL-6 or IL-8 production nor zymosan- or S
aureus-stimulated TNF- In contrast to our results is the finding that exogenous ubiquitin
augments the LPS (1 µg/mL)-stimulated TNF- However, the findings in murine macrophage cell lines as well as the unaffected LPS-evoked IL-6 and IL-8 production by ubiquitin indicate that neutralization of LPS by exogenous ubiquitin, for example, by LPS binding, is not accountable for the inhibitory effects in human PBMNCs. Although exogenous ubiquitin at 100 µg/mL inhibited proliferation in several hematopoietic cell lines after 48 hours of incubation, the inhibitory effect, as measured with the MTT assay, was marginal on MOLT-4 cells and human PBMNCs.10 Therefore, our finding that ubiquitin did not affect viability of human PBMNCs after 4 hours of incubation is not contradictory. Because indirect evidence has been provided for a transport of exogenous ubiquitin into the cell and metabolism via ubiquitylation to target proteins and degradation by the proteasome system,10 a similar mechanism could possibly explain the effects in human PBMNCs. Human PBMNCs were described to contain 50 ng free ubiquitin per cells from 1 mL of blood.26 As estimated from these data, the amount of free ubiquitin approximates 7 fg/cell. In our experiments, the amount of exogenous ubiquitin per PBMNC supplied in the cell cultures was 150- to 300-fold higher. Although the mechanism of ubiquitin transportation into intact cells is unknown, the high extracellular ubiquitin content could possibly explain a significant increase of the intracellular ubiquitin concentration, even if only small proportion of exogenously supplied ubiquitin is transported into the PBMNCs. The ubiquitin serum concentrations determined in healthy volunteers are
in agreement with the normal range determined by
others.7,8,26 Compared with healthy volunteers, ubiquitin
concentrations were found to be significantly (5- to 7-fold) increased
in serum from both trauma and sepsis patients, and to be 4.5-fold
increased in urine from sepsis patients. Surprisingly, patients'
ubiquitin serum concentrations were on a level with the ubiquitin
concentration required for inhibition of the PBMNC TNF- In contrast to IL-10, IL-4, and transforming growth factor- Although comparison of the inhibitory serum activity measured in trauma
and sepsis patients' serum (50% inhibition by 30% [vol/vol] serum
with a mean ubiquitin concentration of 350 ng/mL) with the
dose-dependent effect of exogenous ubiquitin on LPS-evoked TNF- With regard to the higher-molecular-weight bands visualized in serum by
immunoblotting using both antiubiquitin AS and monoclonal UbP4D1
antibody, affinity chromatography showed that they were not bound to
immobilized antiubiquitin AS, whereas free ubiquitin was retained.
Besides low affinity or competitive binding,27 where
sample proteins compete with binding sites and are displaced by
high-affinity bound free ubiquitin, unspecific binding in
immunoblotting could explain that ubiquitin immunoreactive proteins are
detectable in the unadsorbed fractions. However, the finding,
that the unadsorbed fractions exert no effect on the LPS-evoked
TNF- Although antiubiquitin AS was able to neutralize the inhibition induced by sepsis patients' serum on PBMNCs, monoclonal and polyclonal antiubiquitin antibodies showed a decreased neutralizing activity on PBMNCs and hardly any capacity in neutralizing the inhibitory effect of sepsis patients' serum on whole blood cultures. Nevertheless, all used antiubiquitin antibodies were able to
revert reduced LPS-stimulated TNF- Besides the well-described similarity of a reduced TNF- Although the origin of extracellular ubiquitin in critically ill
patients remains to be determined, secretion of intracellularly synthesized ubiquitin10 as well as liberation of
intracellular ubiquitin by tissue damage appears reasonable. In
particular, the latter hypothesis could explain the early appearance of
reduced leukocyte function and availability of inhibitory serum
activity for TNF- In conclusion, the results demonstrate for the first time that extracellular ubiquitin acts as a cytokinelike protein with anti-inflammatory properties and indicate that extracellular ubiquitin is involved in the regulation of immunodepression in critical illness. The relative contribution of extracellular ubiquitin to immunomodulation in various inflammatory situations as well as its biologic significance in vivo remain to be determined.
We thank Mrs Anja Bistron for her excellent technical support.
Submitted March 27, 2002; accepted October 16, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-03-0918.
Supported in part by a grant from the Deutsche Forschungsgemeinschaft (DFG, grant Ma 2474/1-1).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Matthias Majetschak, Department of Trauma Surgery, University Hospital Mannheim, Faculty of Clinical Medicine Mannheim of the Ruprecht-Karls University Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany; e-mail: mmajetschak{at}med.miami.edu.
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© 2003 by The American Society of Hematology.
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  C.-Y. Chen, K.-Y. Yang, M.-Y. Chen, H.-Y. Chen, M.-T. Lin, Y.-C. Lee, R.-P. Perng, S.-L. Hsieh, P.-C. Yang, and T.-Y. Chou Decoy Receptor 3 Levels in Peripheral Blood Predict Outcomes of Acute Respiratory Distress Syndrome Am. J. Respir. Crit. Care Med., October 15, 2009; 180(8): 751 - 760. [Abstract] [Full Text] [PDF]
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 M. Majetschak, U. Krehmeier, L. Ostroverkh, B. Blomeke, and M. Schafer Alterations in Leukocyte Function following Surgical Trauma: Differentiation of Distinct Reaction Types and Association with Tumor Necrosis Factor Gene Polymorphisms Clin. Vaccine Immunol., February 1, 2005; 12(2): 296 - 303. [Abstract] [Full Text] [PDF]
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response to
endotoxin in peripheral blood mononuclear cells and regulates endotoxin
hyporesponsiveness in critical illness
Top
Abstract
Introduction
excitation 485 nm, 
), 500 ng/mL (
), and
1000 ng/mL (
) exogenous ubiquitin. Cultures (in duplicates) were
incubated for 2, 4, 8, and 16 hours. (D) Kinetics of the LPS-stimulated
TNF-
)
whole blood by exogenous ubiquitin. Whole-blood cultures (in
duplicates; n = 3) were incubated for 4 hours with 0 to 1 µg/mL
exogenous ubiquitin in the presence of 100 ng/mL (porcine) and 1 µg/mL (murine) LPS. Data represent means ± SEM.
*P < .05 versus cultures without ubiquitin.
(TGF-
pathway during critical illness.
Blood.
1997;89:1612-1620