Advanced
Interleukin-32γ Transgenic Mice Resist LPS-Mediated Septic Shock
Interleukin-32γ Transgenic Mice Resist LPS-Mediated Septic Shock
Journal of Microbiology and Biotechnology. 2014. Aug, 24(8): 1133-1142
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : April 10, 2014
  • Accepted : April 18, 2014
  • Published : August 30, 2014
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Sun Jong Kim
Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Kunkuk University Hospital, and
Siyoung Lee
Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea
Areum Kwak
Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea
Eunsom Kim
Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea
Seunghyun Jo
Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea
Suyoung Bae
Department of Bioequivalence Division for Drug Evaluation, Ministry of Food and Drug Safety, Chungcheongbuk-do 363-700, Republic of Korea
Youngmin Lee
Department of Medicine, Pusan Paik Hospital, College of Medicine, Inje University, Busan 633-165, Republic of Korea
Soyoon Ryoo
Division of Veterinary Bacterial Disease, and
Jida Choi
Division of Veterinary Epidemiology, Animal and Plant Quarantine Agency, Gyeonggi-do 480-757, Republic of Korea
soohyun@konkuk.ac.kr
Soohyun Kim
Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul 143-701, Republic of Korea
soohyun@konkuk.ac.kr

Abstract
Interleukin-32 (IL-32) is a cytokine and inducer of various proinflammatory cytokines such as TNFα, IL-1β, and IL-6 as well as chemokines. There are five splicing variants (α, β, γ, δ, and ε) and IL-32γ is the most active isoform. We generated human IL-32γ transgenic (IL-32γ TG) mice to express high level of IL-32γ in various tissues, including immune cells. The pathology of sepsis is based on the systemic inflammatory response that is characterized by upregulating inflammatory cytokines in whole body, particularly in response to gram-negative bacteria. We investigated the role of IL-32γ in a mouse model of experimental sepsis by using lipopolysaccharides (LPS). We found that IL-32γTG mice resisted LPS-induced lethal endotoxemia. IL-32γ reduced systemic cytokines release after LPS administration but not the local immune response. IL-32γTG increased neutrophil influx into the initial foci of the primary injected site, and prolonged local cytokines and chemokines production. These results suggest that neutrophil recruitment in IL-32γTG occurred as a result of the local induction of chemokines but not the systemic inflammatory cytokine circulation. Together, our results suggest that IL-32γ enhances an innate immune response against local infection but inhibits the spread of immune responses, leading to systemic immune disorder.
Keywords
Introduction
Interleukin-32 (IL-32) is a cytokine and induces other proinflammatory cytokines, TNFα, IL-1β, and IL-6, as well as chemokines leading to inflammatory disorders [6 , 23 , 25 , 41] . IL-32 has been discovered originally in activated natural killer cells and T cells, however, IL-32 expression is also detected in other immune cells such as macrophages and monocytes [5 , 10 , 16] . Additionally, non-immune cells, including keratinocytes, human umbilical vein endothelial cells, and pancreatic cells, are also sources of IL-32 [27 , 32 , 35] . Although a specific receptor for IL-32 has not been demonstrated, IL-32 has been involved in many inflammatory disorders and infections and its expression has correlated with disease severity [3 , 5 , 19] .
Septic shock is a systemic inflammatory response caused by an overwhelming infection that leads to a serious disorder with high rates of morbidity and mortality, despite the advance of antibiotics and progress in the management of intensive care patients [4] . The most frequent microbial pathogens responsible for septic shock are gram-negative bacteria, which can directly evoke a strong innate immune response in hosts [37] . Lipopolysaccharide (LPS), a main component of gram-negative bacteria outer membranes, binds specifically to Toll-like receptor (TLR)-4 expressed in various immune and non-immune cells and induces a robust immune reaction via induction of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines, including other inflammatory factors [14 , 24 , 39] .
IL-32 consists of several isoforms (α, β, γ, δ, and ε), which show no significant difference in functions and act as a secretary or intracellular protein [2 , 5] . Intracellular IL-32 is upregulated in activated NK and T cells by IFNγ-producing cells and implicated in cell apoptosis [16 , 32] . Endogenous IL-32 also exhibits anti-inflammatory properties by promoting the production of IL-10 and by decreasing the induction of IL-12 in dendritic cells [20] . In contrast, secreted forms of IL-32 act as a proinflammatory cytokine and induce TNFα or IL-8 in monocytes and macrophages derived from human peripheral blood mononuclear cells (PBMCs) and mouse bone marrow cells [2 , 25] .
Sepsis or endotoxemia, the events that follow in the presence of excessive levels of endotoxin due to bacterial overgrowth, consequently lead to failure to maintain a proper balance between excessive and inadequate inflammation and immune responses. [7] . LPS-induced endotoxemia often provokes a life-threatening inflammatory disorder characterized by fever, hypotension, disseminated intravascular coagulation, multiple organ failure, shock, and, in 25-40% of cases, death [30] . This phenomenon can be experimentally reproduced in animals by administration of a high dose of exogenous LPS purified from gram-negative bacteria [15] .
High concentrations of inflammatory cytokines in the blood stream sometimes indicate an increased risk of mortality but targeting to antagonize their activities has only limited success [34 , 38] . Furthermore, mortality is observed in later period of infection and has been ascribed to the second phase of immune suppression that follows the excessive immune responses in the early phase [21] . Increasing evidence suggests that the complex syndrome of sepsis develops when the initial host response is limited to contain the primary infection, resulting in widespread inflammation and organ failure [22] .
As no analog of IL-32 has yet been reported in mice, investigators have assessed the in vivo effect of endogenous IL-32 by generating different isoforms of IL-32 transgenic mice (IL-32TG). Different types of IL-32TG have shown hyperactivation in inflammatory circumstances and, as a result, developed severe clinical signs in the mouse model of sepsis [26 , 33 , 42] . However, our previous study indicated that IL-32γTG mice presented fast and severe development of experimental colitis, but had benefits for recovery and survival [2] . This updated report suggested that IL-32 would have dual effects in a certain immune response in vivo , although IL-32 is originally reported to escalate an inflammation as an inflammatory cytokine. We therefore investigated the role of IL-32γ in experimental sepsis, a complicated inflammatory condition. The present study described here is designed to determine the effect of IL-32γ in experimental sepsis using IL-32γTG and to elucidate the function of IL-32γ to induce immune responses against infection.
Materials and Methods
- Mice
We generated IL-32γTG mice, which overexpressed human IL-32γ, on a C57BL/6 background under a control of a ubiquitous chicken β-actin promoter in the previous study as described [6] . Wild-type (WT) C57BL/6 mice were obtained from Orient (Seoul, Korea). Eight week-old WT and IL-32γTG mice were used for this study. Experimental procedures followed the recommendations for animal use and welfare, as dictated by institutional and national guidelines.
- Induction of Endotoxemia
Endotoxemia was induced in mice by intraperitoneal (i.p.) injection of low dose (3 mg/kg) or high dose (30 mg/kg) of LPS ( E. coli O127:B9; Sigma-Aldrich, St. Louis, MO, USA). All injected mice were closely monitored every 2 to 3 h for the first 16 h and every 12 h thereafter for at least 80 h. In an additional experiment, we collected blood from mice at 1, 4, and 17 h after intraperitoneal injection, and the serum was obtained after clotting for 2 h at 4℃. Samples were centrifuged for 10 min at 12,000 rpm and supernatants were stored at -20℃ until assay.
- Cytokine ELISA
Murine TNFα, IL-6, IL-10, MIP-2, and human IL-32 quantities in samples were determined by standard sandwich ELISA. Development kits of mouse cytokines were used according to manufacturer instructions (R & D systems, Minneapolis, MN, USA). Human IL-32γ concentrations in transgenic mice were determined by a human IL-32 specific sandwich ELISA kit (YbdY, Seoul, Korea). Briefly, 96-well plates were coated with capture antibody overnight at 4℃. A fter blocking f or 1 h with 3 % BSA / PBS, samples and standards were incubated for 2 h at room temperature. The ELISA plates were then incubated with detection antibody for 2 h, and strepavidin/peroxidase for mouse cytokine ELISA or peroxidase-conjugated anti-rabbit IgG was incubated for 45 min. Washing the plate four times with 0.05% Tween/PBS was performed before the addition of all reagents in each procedure. Finally, the color reaction was developed with 3,3’,5,5’-tetramethyl-benzidine liquid substrate (Sigma-Aldrich), and then stopped with 0.1 M H 2 SO 4 . The absorbance at 450 nm was measured using an ELISA plate reader (Molecular Devices, Orleans, CA, USA).
- Bone Marrow-Derived Macrophages Culture
For in vitro studies, macrophages were differentiated from bone marrow cells. Whole bone marrow cells were flushed from femurs and incubated in a differential medium (RPMI 1640, 10% FBS, 30% L929 cell cultured supernatant, 100 U/ml penicillin, and 100 μg/ml streptomycin) for 7 days to allow for proliferation and differentiation. Differentiated bone marrow-derived macrophages (BMDMs) were seeded at a density of 4 × 10 5 cells in 96-well plates for cytokine assays, and in 6-well plates for western blot assay. BMDMs were allowed to adhere for 24 h before use in the assays. Cells were incubated with or without LPS (100 ng/ml) at 37℃ under humidified 5% CO 2 . Supernatants were collected at different time points, centrifuged at 2,000 rpm, and stored at -20℃ until assay of cytokines by ELISA. For detection of phosphorylated NF-κB and p38MAPK, the cells were stimulated as indicated in the Figure legends and lysed directly with RIPA buffer (Cell Signaling, Danvers, MA, USA) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich). Cell lysates were centrifuged at 14,000 rpm for 5 min, and the supernatant was transferred and kept until use for western blot assay.
- Western Blot
For assessing LPS-induced signaling, BMDMs from IL-32γTG and WT were collected after LPS stimulation at different time points. The concentration of total protein was determined using a BCA kit (GE Healthcare, Piscataway, NJ, USA) and 30 μg of protein was resolved on 10% SDS-PAGE gel and transferred to nitrocellulose membranes (GE healthcare). Membranes were blocked in 3% BSA/TBST (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were then incubated with primary antibody for phosphorylated NF-κB and p38MAPK (Cell Signaling), appropriate secondary antibody conjugated to horseradish peroxidase (Cell Signaling), and visualized using ECL plus (GE Healthcare). Signals were detected by a LAS-3000 mini CCD camera (Fuji Film, Tokyo, Japan). The membranes were reprobed with goat anti-IL-32 antibody (YbdY) and then normalized finally with goat anti-actin (Santa Cruz Biotechnology).
- Kupffer Cell Staining
Kupffer cell numbers in the liver were determined by intravenously administering India ink (50 μl) via the tail vein to mice. After 20 min, the livers were removed and fixed in chilled 4% (w/v) paraformaldehyde (Sigma) in PBS, pH 7.4, for tissue section. The fixed tissues were embedded in paraffin blocks, sectioned at 5 μm using a microtome, and placed on glass slides.Tissue sections were deparaffinized with xylene and covered with mounting solution and a cover slide. Kupffer cells in liver sections were quantitated by counting the number of India ink-positive cells in microscopic low-power fields (× 100).
- FACS Staining
Thioglycollate-elicited peritoneal macrophages were isolated from peritoneal cavities after intraperitoneal injections of 3 ml of 3% thioglycollate broth, followed by peritoneal lavage with PBS 4 days later. Peritoneal cells after LPS (30 mg/kg) injection were harvested by peritoneal lavage with 3 ml of PBS at the times indicated in the Figures and legends. Pulmonary macrophages were obtained from broncho-alveolar lavage with PBS. Spleen cells were mechanically disaggregated by mincing the tissues and incubated with hypotonic solution for lysing red blood cells. The cells were washed with PBS, counted, and subjected to FACS analysis. Examination of the cells by flow cytometry was done on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and the data were analyzed on CellQuest software (Becton Dickinson). Isolated cells were washed once in ice-cold PBS containing 2% FBS and 0.1% NaN 3 . Nonspecific binding was blocked by incubation of cells in 10% normal mouse serum/PBS for 15 min, followed by incubation with Gr-1 and F4/80 antibodies conjugated by fluorescence (eBioscience, San Diego, CA, USA) for 30 min. Cells were subsequently washed with PBS containing 2% FBS and 0.1% NaN 3 , resuspended, and analyzed by FACS.
- RT-PCR
Total RNA was isolated with Tri-Reagent (MRC, Canada) from the peritoneal cells. MMLV-RTase (Beams Bio, Korea) converted 2 μg of total RNA to first strand cDNA, and then PCR was performed at 94℃ for 45 sec, 59℃ for 40 sec, and 72℃ for 30 sec for 35 cycles, with chemokine-specific primers and GAPDH primers. Pairs of mouse MCP-1 sense primer, 5’-AGCACCAGC CAACTCTCACT-3’ and reverse primer, 5’-CGGGTCAACTTC ACATT CAAAG-3’; mouse RANTES sense primer, 5’-CTTGCA GAGGACTCTGAGAC-3’ and reverse primer, 5’-ATTTTCCCA GGACCGAGTG-3’; mouse MIP-2 sense primer, 5’-ACACTTCAG CCTAGCGCCAT-3’ and reverse primer, 5’-CAGGTCAGTTAG CCTTGCCT-3’; and GAPDH sense primer, 5’-ACCACAGTCCAT GCCATCAC-3’ and reverse primer, 5’-TCCACCACCCTGTTG CTGTA-3’, were used for the RT-PCR. The PCR products were separated by agarose gel electrophoresis and visualized by ethidium bromide.
- Statistical Analysis
Graphs were created with the GraphPad 4.0 Prism software. All results were expressed as means ± SEM. Survival curves were analyzed by Log-rank (Mantel-Cox) test, and ELISA data were analyzed by the Student t -test, with p < 0.05 as the minimum level of significance.
Results
- IL-32γTG Mice Are Less Sensitive to Endotoxic Shock Induced by LPS Challenge
A high-dose LPS-induced mortality of WT and IL-32γTG was analyzed in Fig. 1 . IL-32γTG mice survived longer than WT mice after LPS administration. In the first 24 h after LPS injection, WT mice succumbed to LPS with a survival rate of 40%, whereas the IL-32γTG group showed high survival rate (86.6%) at the same time point. The difference between IL-32γTG and WT mice was sustained to 42 h of LPS administration as indicated by the asterisk ( Fig. 1 ). There was statistically significant difference between Kaplan-Meier survival curves between IL-32γTG and WT mice, although a final lethality of IL-32γTG was equal to WT mice at the end of the study. This suggests that IL-32γ protects mice from LPS-induced sepsis leading to death.
PPT Slide
Lager Image
The lethality of LPS-induced endotoxin shock in IL-32γTG and WT mice. 32γTG and WT mice were injected with LPS (30 mg/kg) via the intraperitoneal route. All mice were monitored for survival up to 80 h. Kaplan-Meier curve analysis of survival is shown from the cumulation of three experiments (n = 15 in each group). Statistical curve comparison was analyzed by Log-rank test. One of two representative experiments is shown.
- LPS-Mediated Inflammatory Cytokine Productions Decreased in Sera of IL-32γTG Mice
We examined the induction of cytokines by a high-dose LPS (30 mg/kg) injection in sera of WT and IL-32γTG mice. We measured the cytokine level, including TNFα, IL-6 and IL-10, in the sera of LPS-injected mice by a specific ELISA kit. As shown in Fig. 2 , the levels of these cytokines increased in the sera of the WT mice and IL-32γTG mice in response to LPS at different time points. For TNFα, there was an early induction at 1 h after the injection of LPS in both groups, but IL-32γTG produced less TNFα compared with WT, was appeared at 4 and 17 h after LPS administration ( Fig. 2 A). Unlike TNFα, the level of IL-6 increased progressively reaching the maximum at 4 h after LPS administration. The level of IL-6 in IL-32γTG mice was lower than in WT at 17 h ( Fig. 2 B). In addition, we measured the level of the anti-inflammatory cytokine IL-10. Interestingly, the level of IL-10 was also lower in IL-32γTG than in WT mice at 4 and 17 h after LPS administration ( Fig. 2 C).
PPT Slide
Lager Image
IL-32γTG mice exhibit reduced levels of proinflammatory cytokines in the sera after a high-dose LPS administration. WT (C57BL/6) (n = 4) and IL-32γTG (n = 4) were injected i.p. with LPS (30 mg/kg), and sera were collected 0, 1, 4, and 17 h post-injection. The levels of (A) TNFα, (B) IL-6, (C) IL-10, and (D) IL-32γ were assessed by sandwich ELISA as described in Materials and Methods. Student’s t-test *p < 0.05; **p < 0.01. One of three representative experiments is shown.
We also tested the induction of cytokines by a low-dose LPS (3 mg/kg) injection in sera of WT and IL-32γTG mice. As shown in Figs. 3 A and 3 B, the patterns of TNFα and IL-6 production were similar to the high-dose LPS-injected mice, but a difference between IL-32γTG and WT mice was observed with IL-6 production at 1 h after LPS administration ( Fig. 3 B). Interestingly, the low-does LPS-induced IL-10 was observed at 1 h after LPS and the statistical significance was obtained only at 1 h after LPS injection ( Fig. 3 C). We also determined the IL-32 level in sera during LPS endotoxemia in IL-32γTG mice by a specific sandwich ELISA kit. As shown in Figs. 2 D and 3 D, LPS injection influenced the serum IL-32 level in IL-32γTG mice. In the high-dose LPS (30 mg/kg), IL-32γ was slightly decreased until 4 h, and then recovered at 17 h after LPS injection, whereas in the low-dose LPS (3 mg/kg), the IL-32γ level was reduced at 1 h, rebounded at 4 h, and significantly decreased at 17 h after LPS injection ( Fig. 3 D).
PPT Slide
Lager Image
IL-32γTG mice reduced levels of proinflammatory cytokines in the sera after a low-dose LPS administration. WT (C57BL/6) (n = 4) and IL-32γTG mice (n = 4) were injected i.p. with LPS (3 mg/kg), and sera were collected 0, 1, 4, and 17 h post-injection. The levels of (A) TNFα, (B) IL-6, (C) IL-10, and (D) IL-32γ were assessed by sandwich ELISA as described in Materials and Methods. Student’s t-test, ***p < 0.001. One of three representative experiments is shown.
- LPS Responsiveness of Bone Marrow-Derived Macrophages from IL-32γTG and WT Mice
In order to test whether the attenuated response to LPS in IL-32γTG mice is due to an intrinsic defect of target cells, we isolated and cultured BMDMs from WT and IL-32γTG mice in the presence or absence of LPS. The differentiation time and the cell numbers were equal in WT and IL-32γTG bone marrow cells (data not shown). After LPS treatment, supernatants were collected at different time points to measure the levels of MIP-2, TNFα, IL-6, and IL-10. The production of TNFα and IL-10 (data not shown) was not significantly different ( Fig. 4 B). However, MIP-2 was slightly enhanced in IL-32γTG macrophages compared with that of the control WT mice at every time point ( Fig. 4 A) but significance was obtained at 4 and 24 h. We found that at the early time after LPS stimulation, BMDMs of IL-32γTG released higher IL-6 than that of WT at 4 and 9 h, but final production after 24 h was not different between WT and IL-32γTG mice ( Fig. 4 C).
PPT Slide
Lager Image
BMDMs from IL-32γTG mice exhibit equivalent responses to LPS compared with WT. Bone marrow cells collected from WT (n = 3) and IL-32γTG mice (n = 3) were differentiated to macrophages, and cultured in the presence or absence of LPS (100 ng/ml). Supernatants were collected at the indicated time and used for analysis of (A) MIP-2, (B) TNFα, and (C) IL-6 levels by ELISA. Student’s t-test *p < 0.05; **p < 0.01. Representative data are shown of three independent experiments. (D) Activation of the LPS-induced signaling pathway was determined at different time points by detecting the phosphorylated protein levels in the BMDMs isolated from WT and IL-32γTG mice using western blot analysis. One of two representative experiments is shown.
To assess the possibility that LPS signaling may be altered in BMDMs from IL-32γTG, we collected the BMDMs after LPS stimulation and studied the amount of phosphorylated NF-κB p65 (p-NF-κB) and p38MAPK (p-p38MAPK) with protein of BMDMs ( Fig. 4 D). The activity of these signaling molecules induced by LPS reached a peak level at 30 min in BMDMs of IL-32γTG, but at 60 min in BMDMs of WT. In addition, the phosphorylation level of NF-κB in IL-32γTG was weaker than in WT at 60 min ( Fig. 4 D, upper panel). In contrast, the phosphorylation level of p38MAPK in IL-32γTG was higher than that of WT at 30 min ( Fig. 4 D, the second panel).
- Tissue Macrophage Distribution Is Not Influenced by IL-32γTG
Next, we compared the macrophage distribution in the liver, lung, peritoneal cavity, and spleen from WT and IL-32γTG mice. To determine Kupffer cell distribution in the liver, animals were injected intravenously via the tail vein with India ink. Liver sections of WT and IL-32γTG mice revealed that Kupffer cell distribution and its number were similar ( Fig. 5 A). In addition, IL-32γTG mice had normal compositions of alveolar, peritoneal, and splenic macrophages as determined by FACS staining with Gr-1, a neutrophil marker, and F4/80, a macrophage marker, compared with WT ( Figs. 5 B- 5 D). In the steady state of peritoneal lavage, macrophage cells recovered (~2 × 10 6 cells/ per mouse) ~50% equally in both IL-32γTG and WT mice ( Figs. 5 E and 5 F).
PPT Slide
Lager Image
Comparison of macrophage distribution and migration in vivo in WT and IL-32γTG mice. The macrophage proportion in tissue was examined in the liver, lung, peritoneal cavity, and spleen from WT and IL-32γTG mice. (A) The numbers of Kupffer cells were determined by India ink staining as described in Materials and Methods. Representative pictures are shown of three independent experiments. Bars indicate the mean ± SEM. The macrophage percentage in (B) lung, (C) peritoneal cavity, and (D) spleen was analyzed by FACS. Isolated cells from each tissue were stained with Gr-1 and F4/80 Abs conjugated by fluorescence. Macrophage was identified by F4/80 staining alone. (E) The numbers of peritoneal cells and (F) percentage of peritoneal macrophages are presented by graphs. Bars indicate the mean ± SEM.
- Neutrophil Migration into the Primary Injected Site Was Elevated in IL-32γTG
To confirm whether IL-32γ affects immune cell migration following administration of LPS (3 mg/kg), we examined the population of total peritoneal cells in the peritoneal wash fraction labeled with antibodies to Gr-1 and F4/80 from IL-32γTG and WT mice at 2 and 6 h post-injection. As shown in Fig. 6 A, we observed a dramatic increase in the percentage of Gr-1 + cells in IL-32γTG mice compared with WT mice at 2 and 6 h after LPS administration. On the contrary, the populations of F4/80-expressing macrophages were decreased in IL-32γTG mice relatively. To determine whether recruitment of neutrophils to the peritoneal cavity is the result of responses to a chemokine, the major chemoattractant for neutrophil, MIP-2, was checked in the peritoneal lavage and serum by ELISA. The serum level of LPS-induced MIP-2 in IL-32γTG was decreased, as with other proinflammatory cytokines ( Fig. 6 B). In contrast, there was no difference in the amounts of MIP-2 in the peritoneal exudates between IL-32γTG and WT mice after LPS injection. Furthermore, the expressions of several chemokines by RT-PCR were examined in the peritoneal cells from mice after LPS administration. Interestingly, the expressions of MCP-1, RANTES, and MIP-2 were found in IL-32γTG mice, but these ckemokines expressions were absent in WT ( Fig. 6 C). The amounts of MIP-2 and TNFα production in the peritoneal lavage from LPS-injected mice were diminished significantly. Unlike the serum level of cytokines, the cytokines in the peritoneal lavage of IL-32γTG was similar to WT at 2 and 6h post-injection ( Figs. 6 D and 6 E). Interestingly, IL-6 production in the peritoneal cavity of IL-32γTG mice by LPS was sustained longer than in WT mice ( Fig. 6 F). The cytokine profiling of the peritoneal exudates suggests that IL-32γTG elicits active immune responses against LPS locally.
PPT Slide
Lager Image
Analysis of early alteration of neutrophil and macrophage populations in the peritoneal cavity of mice injected with LPS. Peritoneal lavage was performed with WT and IL-32γTG mice at 0, 2, and 6 h after LPS administration, and peritoneal cells were separated by centrifugation. (A) Flow cytometric analysis of Gr-1 and F4/80 staining. Gr-1+ neutrophils, upper left; F4/80+ macrophages, lower right. (B) MIP-2 expression induced in WT and IL-32γTG mice in response to LPS injection. The levels of the neutrophil attracting chemokine MIP-2 in the blood and peritoneal fluid (PF) were determined by ELISA. Data are means ± SEM. *, p < 0.05. (C) Induction of several chemokines in peritoneal cells from WT and IL-32γTG mice by LPS. Expression of chemokines was analyzed using RT-PCR with total RNA isolated in the peritoneal lavage cells at 2 h post-injection. (D) MIP-2, (E) TNFα, and (F) IL-6 production into the peritoneal lavages from WT and IL-32γTG mice by LPS injection. At 2 and 6 h time points, the peritoneal lavages were collected and analyzed by ELISA. Data are shown as means with SEM.
Discussion
In this study, we found that IL-32γTG mice were more resistant to a lethal dose of LPS injected via the intraperitoneal route than WT and achieved long-term survival. Moreover, the levels of serum cytokines, including TNFα, IL-6, and IL-10, in IL-32γTG were lower than in WT mice. Proinflammatory cytokines mediate many of the immune pathological features of septic shock [18] . This is remarkable in severe sepsis, in which the excessive production of proinflammatory cytokines causes capillary leakage, tissue injury, and lethal organ failure [29] . Therefore, the suppression of proinflammatory cytokines has a beneficial effect to reduce the collateral damage and mortality from cytokine overwhelming.
Sepsis arises if a local infection progresses into a systemic inflammatory condition. The failure of an initial and appropriate host response in the inflammed site causes the amplification and dysregulation of inflammatory responses. Neutrophils have a crucial arm in the primary defense mechanism against bacteria invading, as shown by neutropenia such as leukemia and drug-induced neutropenia, which augments susceptibility to infection and to sepsis [8] . Moreover, in some diseases that have a high risk to infection, such as diabetes [28] and acquired immunodeficiency syndrome [31] , an impairment of neutrophil migration is reported. Indeed, IL-32γ dramatically enhanced neutrophil recruitment into the LPS-injected site, which correlated with high and continuous chemokines production by LPS in the peritoneal cavity but not in the blood stream. We detected more chemokine mRNAs in the peritoneal cells of IL-32γTG ( Fig. 6 C). These results indicated that the resident peritoneal cells might be the source of the chemokines in the peritoneal cavity, and IL-32γTG improved the chemokine production in the primary LPS-injected site, which is similar to local infection, but not in the blood stream like systemic circulation. The induction of high levels of chemokines in the peritoneal cavity relative to the blood circulation would establish a chemokine gradient that results in recruiting neutrophils sufficiently into the peritoneal site.
LPS resistance of IL-32γTG mice is mainly due to their impaired cytokine production. Mononuclear cells play a key role, releasing the classic proinflammatory cytokines IL-1, IL-6, and TNFα, and macrophages are the first targets of LPS [17] . However, macrophages from IL-32γTG mice responded to LPS normally and settled in tissues properly compared with WT mice. Therefore, the resistance of IL-32γTG mice to LPS could be related to their ability to block the process of LPS-induced systemic immune response rather than a direct result of their low induction of inflammatory factors. Cytokine production in the peritoneal cavity of IL-32γTG mice was also not reduced compared with controls and, even in case of IL-6, sustained higher than WT mice ( Fig. 6 F). The results indicate that the immune responses in the initial site would be strongly elicited in IL-32γTG mice contrary to the entire body. Consequently, our data suggest that IL-32γ could attenuate widespread inflammation by enhancing chemokine-mediated neutrophil influx and production of proinflammatory cytokines in the primary infectious foci.
Experimental sepsis models have provided the knowledge of specific inflammatory mediators and adhesion molecules implicated in sepsis, but conclusions so far have yet to have clinical benefits. Antibody therapies against TNFα and IFNγ protect baboons [40] and mice [11] from bacterial infection, but antagonizing TNFα is ineffective in humans with sepsis [1 , 9] and is even harmful [13] . Similarly, the supplementation of IFNγ enhances neutrophil function in several infectious models of mice [12] but does not improve patients’ outcomes [36] . A fact commonly ignored is that, in humans, neutrophils are the major population of leukocytes in the blood, whereas in the mouse, usually used as an animal model of sepsis, neutrophils are a minor population. Current treatment trials based on the previously established pathology are inadequate to reduce the high mortality rate.
In conclusion, these present data suggested that LPS-induced immune responses were suppressed in IL-32γTG mice by restraining systemic inflammation, but local immune responses were not affected in the primary injected area. Our findings on the mechanism of IL-32γ in the LPS-induced mouse model of septic shock will provide new insights on the role of IL-32γ in sepsis and other inflammatory diseases.
Acknowledgements
This paper was supported by Konkuk University in 2013.
References
Abraham E , Wunderink R , Silverman H , Perl TM , Nasraway S , Levy H 1995 Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, doubleblind, multicenter clinical trial. JAMA TNF-alpha MAb Sepsis Study Group 273 934 - 941    DOI : 10.1001/jama.1995.03520360048038
Alves-Filho JC , Freitas A , Souto FO , Spiller F , Paula-Neto H , Silva JS 2009 Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis. Proc. Natl. Acad. Sci. USA 106 4018 - 4023    DOI : 10.1073/pnas.0900196106
Alves-Filho JC , Sonego F , Souto FO , Freitas A , Verri WA , Auxiliadora-Martins M 2010 Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection. Nat. Med. 16 708 - 712    DOI : 10.1038/nm.2156
Astiz ME , Rackow EC 1998 Septic shock. Lancet 351 1501 - 1505    DOI : 10.1016/S0140-6736(98)01134-9
Bae S , Kim YG , Choi J , Hong J , Lee S , Kang T 2012 Elevated interleukin-32 expression in granulomatosis with polyangiitis. Rheumatology (Oxford) 51 1979 - 1988    DOI : 10.1093/rheumatology/kes163
Choi J , Bae S , Hong J , Ryoo S , Jhun H , Hong K 2010 Paradoxical effects of constitutive human IL-32{gamma} in transgenic mice during experimental colitis. Proc. Natl. Acad. Sci. USA 107 21082 - 21086    DOI : 10.1073/pnas.1015418107
Choi JD , Bae SY , Hong JW , Azam T , Dinarello CA , Her E 2009 Identification of the most active interleukin-32 isoform. Immunology 126 535 - 542    DOI : 10.1111/j.1365-2567.2008.02917.x
Cohen J 2002 The immunopathogenesis of sepsis. Nature 420 885 - 891    DOI : 10.1038/nature01326
Cohen J , Carlet J 1996 INTERSEPT: an international, multicenter, placebo-controlled trial of monoclonal antibody to human tumor necrosis factor-alpha in patients with sepsis. Crit. Care Med. International Sepsis Trial Study Group 24 1431 - 1440    DOI : 10.1097/00003246-199609000-00002
Dahl CA , Schall RP , He HL , Cairns JS 1992 Identification of a novel gene expressed in activated natural killer cells and T cells. J. Immunol. 148 597 - 603
Doherty GM , Lange JR , Langstein HN , Alexander HR , Buresh CM , Norton JA 1992 Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha. J. Immunol. 149 1666 - 1670
Ellis TN , Beaman BL 2004 Interferon-gamma a ctivation of polymorphonuclear neutrophil function. Immunology 112 2 - 12    DOI : 10.1111/j.1365-2567.2004.01849.x
Fisher CJ , Agosti JM , Opal SM , Lowry SF , Balk RA , Sadoff JC 1996 Treatment of septic shock with the tumor necrosis factor receptor:Fc fusion protein. N. Engl. J. Med. The Soluble TNF Receptor Sepsis Study Group 334 1697 - 1702    DOI : 10.1056/NEJM199606273342603
Fitzgerald KA , Rowe DC , Golenbock DT 2004 Endotoxin recognition and signal transduction by the TLR4/MD2-complex. Microbes Infect. 6 1361 - 1367    DOI : 10.1016/j.micinf.2004.08.015
Freise H , Bruckner UB , Spiegel HU 2001 Animal models of sepsis. J. Invest. Surg. 14 195 - 212    DOI : 10.1080/089419301750420232
Goda C , Kanaji T , Kanaji S , Tanaka G , Arima K , Ohno S , Izuhara K 2006 Involvement of IL-32 in activation-induced cell death in T cells. Int. Immunol. 18 233 - 240    DOI : 10.1093/intimm/dxh339
Guha M , Mackman N 2001 LPS induction of gene expression in human monocytes. Cell Signal. 13 85 - 94    DOI : 10.1016/S0898-6568(00)00149-2
Hack CE , Aarden LA , Thijs LG 1997 Role of cytokines in sepsis. Adv. Immunol. 66 101 - 195
Heinhuis B , Koenders MI , van Riel PL , van de Loo FA , Dinarello CA , Netea MG 2010 Tumour necrosis factor alpha-driven IL-32 expression in rheumatoid arthritis synovial tissue amplifies an inflammatory cascade. Ann. Rheum. Dis. 70 660 - 667    DOI : 10.1136/ard.2010.139196
Hong J , Bae S , Kang Y , Yoon D , Bai X , Chan ED 2009 Suppressing IL-32 in monocytes impairs the induction of the proinflammatory cytokines TNFalpha and IL-1beta. Cytokine 49 171 - 176    DOI : 10.1016/j.cyto.2009.10.003
Hotchkiss RS , Coopersmith CM , McDunn JE , Ferguson TA 2009 The sepsis seesaw: tilting toward immunosuppression. Nat. Med. 15 496 - 497    DOI : 10.1038/nm0509-496
Hotchkiss RS , Karl IE 2003 The pathophysiology and treatment of sepsis. N. Engl. J. Med. 348 138 - 150    DOI : 10.1056/NEJMra021333
Joosten LA , Netea MG , Kim SH , Yoon DY , Oppers-Walgreen B , Radstake TR 2006 IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 103 3298 - 3303    DOI : 10.1073/pnas.0511233103
Kawai T , Akira S 2010 The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11 373 - 384    DOI : 10.1038/ni.1863
Kim SH , Han SY , Azam T , Yoon DY , Dinarello CA 2005 Interleukin-32: a cytokine and inducer of TNFalpha. Immunity 22 131 - 142
Kobayashi H , Huang J , Ye F , Shyr Y , Blackwell TS , Lin PC 2010 Interleukin-32beta propagates vascular inflammation and exacerbates sepsis in a mouse model. PLoS One 5 e9458 -    DOI : 10.1371/journal.pone.0009458
Kobayashi H , Yazlovitskaya EM , Lin PC 2009 Interleukin-32 positively regulates radiation-induced vascular inflammation. Int. J. Radiat. Oncol. Biol. Phys. 74 1573 - 1579    DOI : 10.1016/j.ijrobp.2009.04.017
Koh GC , Peacock SJ , van der Poll T , Wiersinga WJ 2011 The impact of diabetes on the pathogenesis of sepsis. Eur. J. Clin. Microbiol. Infect. Dis. 31 379 - 388    DOI : 10.1007/s10096-011-1337-4
Marshall JC , Cook DJ , Christou NV , Bernard GR , Sprung CL , Sibbald WJ 1995 Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome. Crit. Care Med. 23 1638 - 1652    DOI : 10.1097/00003246-199510000-00007
Martin GS , Mannino DM , Eaton S , Moss M 2003 The epidemiology of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 348 1546 - 1554    DOI : 10.1056/NEJMoa022139
Mastroianni CM , Lichtner M , Mengoni F , D’Agostino C , Forcina G , d’Ettorre G 1999 Improvement in neutrophil and monocyte function during highly active antiretroviral treatment of HIV-1-infected patients. AIDS 13 883 - 890    DOI : 10.1097/00002030-199905280-00003
Meyer N , Zimmermann M , Burgler S , Bassin C , Woehrl S , Moritz K 2010 IL-32 is expressed by human primary keratinocytes and modulates keratinocyte apoptosis in atopic dermatitis. J. Allergy Clin. Immunol. 125 858 - 865    DOI : 10.1016/j.jaci.2010.01.016
Nakayama M , Niki Y , Kawasaki T , Takeda Y , Horiuchi K , Sasaki A 2012 Enhanced susceptibility to lipopolysaccharideinduced arthritis and endotoxin shock in interleukin-32 alpha transgenic mice through induction of tumor necrosis factor alpha. Arthritis Res. Ther. 14 R120 -    DOI : 10.1186/ar3850
Netea MG , van der Meer JW , van Deuren M , Kullberg BJ 2003 Proinflammatory cytokines and sepsis syndrome: not enough, or too much of a good thing? Trends Immunol. 24 254 - 258    DOI : 10.1016/S1471-4906(03)00079-6
Nishida A , Andoh A , Inatomi O , Fujiyama Y 2009 Interleukin-32 expression in the pancreas. J. Biol. Chem. 284 17868 - 17876    DOI : 10.1074/jbc.M900368200
Polk HC , Cheadle WG , Livingston DH , Rodriguez JL , Starko KM , Izu AE 1992 A randomized prospective clinical trial to determine the efficacy of interferon-gamma in severely injured patients. Am. J. Surg. 163 191 - 196    DOI : 10.1016/0002-9610(92)90099-D
Remick DG 2007 Pathophysiology of sepsis. Am. J. Pathol. 170 1435 - 1444    DOI : 10.2353/ajpath.2007.060872
Riedemann NC , Guo RF , Ward PA 2003 Novel strategies for the treatment of sepsis. Nat. Med. 9 517 - 524    DOI : 10.1038/nm0503-517
Ryoo S , Lee S , Jo S , Kwak A , Kim E , Lee J 2014 Effect of lipopolysaccharide (LPS) on mouse model of steroidinduced avascular necrosis in the femoral head (ANFH). J. Microbiol. Biotechnol. 24 394 - 400    DOI : 10.4014/jmb.1311.11057
Schlag G , Redl H , Davies J , Haller I 1994 Anti-tumor necrosis factor antibody treatment of recurrent bacteremia in a baboon model. Shock discussion 19-22 2 10 - 18    DOI : 10.1097/00024382-199407000-00002
Shioya M , Nishida A , Yagi Y , Ogawa A , Tsujikawa T , Kim-Mitsuyama S 2007 Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease. Clin. Exp. Immunol. 149 480 - 486    DOI : 10.1111/j.1365-2249.2007.03439.x
Shoda H , Fujio K , Yamaguchi Y , Okamoto A , Sawada T , Kochi Y , Yamamoto K 2006 Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases. Arthritis Res. Ther. 8 R166 -    DOI : 10.1186/ar2074