The beta-glucan receptor,dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages

We recently identified dectin-1 (betaGR) as a major beta-glucan receptor on leukocytes and demonstrated that it played a significant role in the non-opsonic recognition of soluble and particulate beta-glucans. Using a novel mAb (2A11) raised against betaGR, we show here that the receptor is not dendritic cell-restricted as first reported, but is broadly expressed, with highest surface expression on populations of myeloid cells (monocyte/macrophage (Mphi) and neutrophil lineages). Dendritic cells and a subpopulation of T cells also expressed the betaGR, but at lower levels. Alveolar Mphi, like inflammatory Mphi, exhibited the highest surface expression of betaGR, indicative of a role for this receptor in immune surveillance. In contrast, resident peritoneal Mphi expressed much lower levels of betaGR on the cell surface. Characterization of the nonopsonic recognition of zymosan by resident peritoneal Mphi suggested the existence of an additional beta-glucan-independent mechanism of zymosan binding that was not observed on elicited or bone marrow-derived Mphi. Although this recognition could be inhibited by mannan, we were able to exclude involvement of the Mphi mannose receptor and complement receptor 3 in this process. These observations imply the existence of an additional mannan-dependent receptor involved in the recognition of zymosan by resident peritoneal Mphi.     

Expression of functionally different dectin-1 isoforms by murine macrophages

Dectin-1 is a specific receptor for beta-glucans and a major receptor for fungal particles on macrophages (Mphi). It is a type II membrane receptor that has a C-terminal, NK-like, C-type lectin-like domain separated from the cell membrane by a short stalk region and a cytoplasmic immunoreceptor tyrosine-based activation-like motif. We observed functional differences in dectin-1-dependent recognition of fungal particles by Mphi from different mouse strains. RT-PCR analysis revealed that mice have at least two splice forms of dectin-1, generated by differential usage of exon 3, encoding the full-length dectin-1A and a stalkless Mphi dectin-1B. Mphi from BALB/c mice and genetically related mice expressed both isoforms in similar amounts, whereas Mphi from C57BL/6 and related mice mainly expressed the smaller isoform. NIH-3T3 fibroblast and RAW264.7 macrophage cell lines stably expressing either isoform were able to bind and phagocytose zymosan at 37 degrees C. However, binding by the smaller dectin-1B isoform was significantly affected at lower temperatures. These properties were shared by the equivalent human isoforms. The relative ability of each of the isoforms to induce TNF-alpha production in RAW264.7 Mphi was also found to be different. These results are the first evidence that dectin-1 isoforms are functionally distinct and indicate that differential isoform usage may represent a mechanism of regulating cellular responses to beta-glucans.     

Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide

Dectin-1 is the major macrophage receptor for beta-glucans and generates a proinflammatory response through the recognition of these carbohydrates on fungal pathogens. We have examined the effects of cytokines and other agents on the expression and functions of dectin-1 in both resident and elicited murine peritoneal macrophages (Mphi). Dectin-1 expression was found to be highly up-regulated by GM-CSF and by the cytokines that induce alternative macrophage activation, IL-4 and IL-13. In contrast, IL-10, LPS, and dexamethasone, but not IFN-gamma, down-regulated the expression of this receptor. Modulation of dectin-1 receptor levels correlated with the ability of these macrophages to bind zymosan and significantly affected the contribution of this receptor to the resultant proinflammatory response, as measured by the production of TNF-alpha, although some Mphi-specific differences were observed. These results correlate with the known effects of these cytokines and other agents on the ability of the immune system to recognize and respond to fungal pathogens.     

Enhanced defense against Pneumocystis carinii mediated by a novel dectin-1 receptor Fc fusion protein

Pneumocystis carinii (PC) pneumonia is a leading opportunistic infection found among HIV-infected individuals worldwide. Although CD4(+) T cell deficiency clearly correlates with susceptibility to PC pneumonia, murine models of disease indicate that PC-directed Abs may prevent infection and/or inhibit growth of existing PC within the lungs. Recognition of PC by alveolar macrophages involves the beta-glucan receptor Dectin-1 and macrophage effector function against PC is enhanced by Abs derived from PC-vaccinated hosts. We developed a fusion protein consisting of the extracellular domain of Dectin-1 linked to the Fc portion of murine IgG1, which we hypothesized would enhance host recognition and opsonic phagocytosis of PC. The recombinant protein, Dectin-Fc, is dimeric and the Ag recognition site identifies beta-1,3 glucan linkages specifically and with high affinity (K(D) = 2.03 x 10(-7) M). Dectin-Fc enhances RAW264.7 macrophage recognition of the beta-glucan containing particulate zymosan in an FcgammaRII- and FcgammaRIII-dependent manner and preopsonization of PC organisms with Dectin-Fc increased alveolar and peritoneal macrophage-dependent killing of PC. SCID mice treated with a replication incompetent adenoviral vector expressing Dectin-Fc had attenuated growth of PC within the lungs, overall decreased PC lung burden, and diminished correlates of PC-related lung damage relative to SCID mice receiving a control vector. These findings demonstrate that targeting PC beta-glucan with Dectin-Fc enhances host recognition and clearance of PC in the absence of B and T cells, and suggest that FcgammaR-based targeting of PC, via cell wall carbohydrate recognition, may promote resistance against PC pneumonia in the immunodeficient host.     

Translational event mediates differential production of tumor necrosis factor-alpha in hyaluronan-stimulated microglia and macrophages

Recent evidence has demonstrated that hyaluronan synthase 2 mRNA is up-regulated after brain ischemia. After a cerebral ischemic event, microglia and macrophages are the major inflammatory cells and are activated by hyaluronan (HA). However, it is unclear how these cells compare with regard to HA responsiveness. We show here that peritoneal macrophages and RAW 264.7 macrophages produced more than five- and 10-fold more tumor necrosis factor-alpha (TNF-alpha) than primary microglia and BV-2 microglia, respectively. Antibody blockade study showed that CD44, Toll-like receptor-4 receptor and the receptor for HA-mediated motility were responsible for HA-induced TNF-alpha release. Furthermore, HA induced higher levels of phosphorylated MAPK in RAW 264.7 cells when compared with BV-2 cells. HA-mediated TNF-alpha production required p38 MAPK, extracellular-regulated kinase and c-Jun N-terminal kinase phosphorylation in both cell types. The levels of HA-induced TNF-alpha mRNA expression in BV-2 cells were only twofold lower compared with RAW 264.7 cells, suggesting that a translational event is involved in the differential production of TNF-alpha. Western blot analysis revealed that HA treatment resulted in more rapid phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and more effective dissociation of 4E-BP1 from eukaryotic initiation factor 4E in RAW 264.7 cells than in BV-2 cells. Additionally, HA-induced phosphorylation of 4E-BP1 was dependent on MAPK signaling, indicating that RAW 264.7 cells exhibited higher levels of hyperphosphorylated 4E-BP1 possibly due to the overactivation of MAPK. The results suggest that resident microglia and blood-derived monocytes/macrophages exhibit differential sensitivities in response to extracellular mediators after brain ischemia.     

In vitro and in vivo induction of heme oxygenase 1 in mouse macrophages following melanocortin receptor activation

RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4-24 h) and concentration-related (1-100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (Mphi) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary Mphi. HO-1 induction in peritoneal Mphi was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the Mphi, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.     

Regulation of cytosolic phospholipase A2 activation and cyclooxygenase 2 expression in macrophages by the beta-glucan receptor

Phagocytosis of non-opsonized microorganisms by macrophages initiates innate immune responses for host defense against infection. Cytosolic phospholipase A(2) is activated during phagocytosis, releasing arachidonic acid for production of eicosanoids, which initiate acute inflammation. Our objective was to identify pattern recognition receptors that stimulate arachidonic acid release and cyclooxygenase 2 (COX2) expression in macrophages by pathogenic yeast and yeast cell walls. Zymosan- and Candida albicans-stimulated arachidonic acid release from resident mouse peritoneal macrophages was blocked by soluble glucan phosphate. In RAW264.7 cells arachidonic acid release, COX2 expression, and prostaglandin production were enhanced by overexpressing the beta-glucan receptor, dectin-1, but not dectin-1 lacking the cytoplasmic tail. Pure particulate (1, 3)-beta-D-glucan stimulated arachidonic acid release and COX2 expression, which were augmented in a Toll-like receptor 2 (TLR2)-dependent manner by macrophage-activating lipopeptide-2. However, arachidonic acid release and leukotriene C(4) production stimulated by zymosan and C. albicans were TLR2-independent, whereas COX2 expression and prostaglandin production were partially blunted in TLR2(-/-) macrophages. Inhibition of Syk tyrosine kinase blocked arachidonic acid release and COX2 expression in response to zymosan, C. albicans, and particulate (1, 3)-beta-D-glucan. The results suggest that cytosolic phospholipase A(2) activation triggered by the beta-glucan component of yeast is dependent on the immunoreceptor tyrosine-based activation motif-like domain of dectin-1 and activation of Syk kinase, whereas both TLR2 and Syk kinase regulate COX2 expression.     

Cross-linking of SIGNR1 activates JNK and induces TNF-α production in RAW264.7 cells that express SIGNR1

In this study, we evaluated the signaling ability of SIGNR1 in murine macrophage-like RAW264.7 cells that stably expressed FLAG-tagged SIGNR1 (SIGNR1-FLAG). Cross-linking of SIGNR1-FLAG expressed on the cells by an anti-FLAG antibody induced JNK phosphorylation without induction of phosphorylation of ERK1/2 and p38 MAP kinase, and led to phosphorylations of Src family kinases (SFKs) and Akt. The SIGNR1-FLAG molecules in the cells were found in lipid raft-enriched membrane fractions, and the tyrosine kinases Lyn, Hck, and Fgr co-precipitated with SIGNR1-FLAG in the lipid raft fractions. The antibody-induced JNK phosphorylation was inhibited by inhibitors of SFKs and tyrosine kinases. Furthermore, cross-linking of SIGNR1 led to production of TNF-α, and the JNK inhibitor inhibited the antibody-induced TNF-α production. These results show that cross-linking of SIGNR1 triggers phosphorylation of SFKs, which leads to activation of the JNK pathway and induction of TNF-α production in macrophage-like RAW264.7 cells.     

Imidocarb,a potent anti-protozoan drug,up-regulates interleukin-10 production by murine macrophages

Interleukin-10 (IL-10), a potent antiinflammatory and immunosuppressive cytokine, plays an important role in the regulation of immune responses. To discover small molecules that stimulate IL-10 production, a cell-based screening assay was performed using a murine macrophage cell line, RAW264.7. Imidocarb, (3,3'-bis-2-imidazolin-2-yl)-carbanilide, which has been used as an anti-protozoan drug for the prevention and treatment of babesiosis in cattle, was thus identified. Imidocarb markedly enhanced LPS-induced IL-10 production not only by RAW264.7 cells but also by murine peritoneal macrophages in a concentration-dependent manner. It also augmented IL-10 production in the presence of zymosan, a yeast cell wall component. In contrast, imidocarb inhibited LPS-induced tumor necrosis factor (TNF)-alpha production by peritoneal macrophages. In mice, intraperitoneal administration of imidocarb significantly increased serum IL-10 levels and lowered TNF-alpha levels. Our results suggest that a novel anti-inflammatory property of imidocarb could lead to new therapeutic approaches in inflammatory conditions.     

Conditional macrophage ablation demonstrates that resident macrophages initiate acute peritoneal inflammation

The role played by resident macrophages (Mphi) in the initiation of peritoneal inflammation is currently unclear. We have used a conditional Mphi ablation strategy to determine the role of resident peritoneal Mphi in the regulation of neutrophil (PMN) recruitment in experimental peritonitis. We developed a novel conditional Mphi ablation transgenic mouse (designated CD11bDTR) based upon CD11b promoter-mediated expression of the human diphtheria toxin (DT) receptor. The murine DT receptor binds DT poorly such that expression of the human receptor confers toxin sensitivity. Intraperitoneal injection of minute (nanogram) doses of DT results in rapid and marked ablation of F4/80-positive Mphi populations in the peritoneum as well as the kidney, and ovary. In experimental peritonitis, resident Mphi ablation resulted in a dramatic attenuation of PMN infiltration that was rescued by the adoptive transfer of resident nontransgenic Mphi. Attenuation of PMN infiltration was associated with diminished CXC chemokine production at 1 h. These studies indicate a key role for resident peritoneal Mphi in sensing perturbation to the peritoneal microenvironment and regulating PMN infiltration.     

Identification of a novel inhibitor of LPS-induced TNF-alpha production with antiproliferative activity in monocyte/macrophages

An isoquinoline derivative, 5-methyl-7,8-dimethoxy-1-phenylpyrazolo[5,4-c]isoquinoline (compound 1), was identified as a novel inhibitor of LPS-induced TNF-alpha production by cell-based screening. Compound 1 suppressed LPS-induced TNF-alpha production in RAW264.7 cells and murine peritoneal macrophages in a dose-dependent manner similar to SB203580, known as a specific inhibitor of p38 MAPK. It also inhibited an LPS-induced increase in serum TNF-alpha in a mouse endotoxic shock model with an ED(50) of approximately 10 mg/kg. Compound 1 had little effect on the incorporation of [3H]-leucine into the cells, while it suppressed LPS-induced TNF-alpha mRNA levels in RAW264.7 cells. The results indicate that suppression of TNF-alpha production was not a result of nonspecific inhibition of de novo translation but was based on the decreased TNF-alpha mRNA levels. The in vitro kinase assay revealed that compound 1 did not strongly inhibit p38 MAPK activity, its potency being much lower than that of SB203580, suggesting that the TNF-alpha-suppressive action of compound 1 cannot be attributed to the inhibition of p38 MAPK. Furthermore, in contrast to SB203580, it significantly inhibited the growth of RAW264.7 and THP-1 cells in a cytostatic manner. Compound 1 is likely to have antiinflammatory and antiproliferative effects by acting on some molecule other than p38 MAPK that contributes to both LPS-induced TNF-alpha production and the cell growth of monocyte/macrophages.     

Molecular mechanism of tumor necrosis factor-alpha production in 1-->3-beta-glucan (zymosan)-activated macrophages

The molecular details of 1-->3-beta-glucans, a fungal cell wall component, induced inflammatory responses are not well understood. In the present study, we conducted a systematic analysis of the molecular events leading to tumor necrosis factor (TNF)-alpha production after glucan stimulation of macrophages. We demonstrated that activation of nuclear factor kappaB (NF-kappaB) is essential in zymosan A (a source of 1-->3-beta-glucans)-induced TNF-alpha production in macrophages (RAW264.7 cells). Zymosan A-induced TNF-alpha protein production was associated with an increase in the TNF-alpha gene promoter activity. Activation of the TNF-alpha gene promoter was dependent on activation of NF-kappaB. Time course studies indicated that DNA binding activity of NF-kappaB preceded TNF-alpha promoter activity. Inhibition of NF-kappaB activation led to a dramatic reduction in both TNF-alpha promoter activity and TNF-alpha protein production in the response to zymosan A. Mutation of a major NF-kappaB binding site (kappa3) in the gene promoter resulted in a significant decrease in the induction of the gene promoter by zymosan A, while mutation of Egr or CRE sites failed to inhibit the response to zymosan. Together, these results strongly suggest that NF-kappaB is involved in signal transduction of 1-->3-beta-glucans-induced TNF-alpha expression.     

RAW264.7 cells lack prostaglandin-dependent autoregulation of tumor necrosis factor-alpha secretion

Studies of the response of RAW264.7 cells (RAW) to lipopolysaccharide (LPS) were carried out to determine why these cells do not demonstrate the prostaglandin (PG)-dependent autocrine regulation of tumor necrosis factor-alpha (TNF-alpha) secretion observed in primary resident peritoneal macrophages (RPMs). The major cyclooxygenase (COX) product of LPS-stimulated RAW was PGD2, with lesser amounts of PGE2. LPS-treated RAW produced PGs more slowly and reached their maximal PG synthetic rate later than did LPS-treated RPMs, as a result of lower constitutive COX-1 expression and a slower rate of COX-2 induction. Cytosolic phospholipase A2 and levels of free arachidonic acid were similar in RAW and RPMs. In contrast to RPMs, LPS-treated RAW produced high quantities of TNF-alpha, which were not altered in the presence of COX inhibitors. This failure of endogenous PGs to suppress TNF-alpha secretion was explained by the absence of the prostaglandin D2 receptor and the low levels of PGE2 produced during the first 2 h of the LPS response. These studies demonstrate that autocrine regulation of TNF-alpha secretion in response to LPS is greatly facilitated by a COX-1-mediated rapid accumulation of PGs as well by a correspondence between the PGs produced and the receptors expressed by the cells.     

稳定表达鼠源PLEKHA1细胞系的建立及其对细胞迁移的影响

目的：构建稳定表达鼠源PLEKHA1的RAW264.7小鼠巨噬细胞系,探讨PLEKHA1过表达对巨噬细胞迁移的影响。方法：根据小鼠PLEKHA1基因序列设计引物,克隆其编码区序列,酶切后插入pCDH载体,在293FT细胞中进行病毒的包装,用获得的高滴度慢病毒感染RAW264.7细胞,建立能稳定高效表达PLEKHA1的RAW264.7细胞系;在此基础上,观察PLEKHA1对巨噬细胞迁移的影响。结果：经基因克隆、酶切、连接后,构建了鼠源PLEKHA1重组慢病毒表达载体,包装病毒感染RAW264.7细胞,经嘌呤霉素筛选后免疫印迹检测,RAW264.7细胞中PLEKHA1的蛋白表达提高近30倍;同时,发现PLEKHA1的过表达影响RAW264.7细胞的迁移。结论：构建了稳定表达小鼠PLEKHA1的RAW264.7细胞系,PLEKHA1过表达降低RAW264.7细胞的迁移能力。     

Differential effects of a Toll-like receptor antagonist on Mycobacterium tuberculosis-induced macrophage responses

We previously showed that viable Mycobacterium tuberculosis (Mtb) bacilli contain distinct ligands that activate cells via the mammalian Toll-like receptor (TLR) proteins TLR2 and TLR4. We now demonstrate that expression of a dominant negative TLR2 or TLR4 proteins in RAW 264.7 macrophages partially blocked Mtb-induced NF-kappa B activation. Coexpression of both dominant negative proteins blocked virtually all Mtb-induced NF-kappa B activation. The role of the TLR4 coreceptor MD-2 was also examined. Unlike LPS, Mtb-induced macrophage activation was not augmented by overexpression of ectopic MD-2. Moreover, cells expressing an LPS-unresponsive MD-2 mutant responded normally to Mtb. We also observed that the lipid A-like antagonist E5531 specifically inhibited TLR4-dependent Mtb-induced cellular responses. E5531 could substantially block LPS- and Mtb-induced TNF-alpha production in both RAW 264.7 cells and primary human alveolar macrophages (AM phi). E5531 inhibited Mtb-induced AM phi apoptosis in vitro, an effect that was a consequence of the inhibition of TNF-alpha production by E5531. In contrast, E5531 did not inhibit Mtb-induced NO production in RAW 264.7 cells and AM phi. Mtb-stimulated peritoneal macrophages from TLR2- and TLR4-deficient animals produced similar amounts of NO compared with control animals, demonstrating that these TLR proteins are not required for Mtb-induced NO production. Lastly, we demonstrated that a dominant negative MyD88 mutant could block Mtb-induced activation of the TNF-alpha promoter, but not the inducible NO synthase promoter, in murine macrophages. Together, these data suggest that Mtb-induced TNF-alpha production is largely dependent on TLR signaling. In contrast, Mtb-induced NO production may be either TLR independent or mediated by TLR proteins in a MyD88-independent manner.     

5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.     

Hydrogen peroxide induces the production of tumor necrosis factor-alpha in RAW 264.7 macrophage cells via activation of p38 and stress-activated protein kinase

The effect of hydrogen peroxide (H(2)O(2)) on production of tumor necrosis factor (TNF)-alpha was examined in RAW 264.7 murine macrophage cells. H(2)O( 2) led to production of TNF-alpha up to 24 h after the treatment, but not nitric oxide in RAW 264.7 cells. H(2)O(2) induced TNF-alpha production in mouse peritoneal macrophages as well as RAW 264.7 cells. The H(2)O(2)induced TNF-alpha production was prevented by inhibitors of p38 and stress-activated protein kinase (SAPK/JNK), and H(2)O( 2) induced the phosphorylation of p38 and SAPK. Further, H(2)O( 2) significantly augmented the AP-1 activity, but not nuclear factor (NF)-kappaB activity in RAW 264.7 cells. A high level of intracellular reactive oxygen radicals (ROS) was detected in H(2)O(2)-exposed RAW 264.7 cells. Ebselen, a cell permeable antioxidant, prevented the H( 2)O(2)-induced TNFalpha production. H(2)O(2) significantly enhanced lipopolysaccharide (LPS)-induced TNF-alpha production. Therefore, H( 2) O(2) was suggested to induce TNF-alpha production in macrophages via activating p38 and SAPK/JNK as oxidative stress-related signal pathways.     

Calcium/calmodulin-dependent protein kinase (CaMK) IV mediates nucleocytoplasmic shuttling and release of HMGB1 during lipopolysaccharide stimulation of macrophages

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mphi) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mphi were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mphi isolated from CaMKIV(+/+) and CaMKIV(-/-) mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mphi treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV(+/+) Mphi showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV(-/-) Mphi. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.