Age-related dysfunction of p53-regulated phagocytic activity in macrophages.

Aging promotes polarization of M2-like macrophages to M1-like macrophages and reduces their phagocytic ability. However, the molecular mechanisms underlying these aging-related changes remain poorly understood. Here, we demonstrate that p53 regulates phagocytic activity in macrophages from young mice but not in those from old ones. Macrophages from both old and young mice expressed functional p53 to induce target genes including p21 and Mdm2. In macrophages from young mice, chemically induced p53 decreased phagocytic activity and c-Myc levels, with the latter change reducing M2-related genes. However, in macrophages from old mice, phagocytic activity and c-Myc expression were independent of p53 activity. Furthermore, c-Myc suppression did not affect M2-related genes in old-mouse macrophages. These results demonstrate that dysregulation of p53 function is a molecular mechanism underlying reduced phagocytic activity in aged-mouse macrophages.

PHLDA1, another PHLDA family protein that inhibits Akt.

The PHLDA family (pleckstrin homology-like domain family) of genes consists of 3 members: PHLDA1, 2, and 3. Both PHLDA3 and PHLDA2 are phosphatidylinositol (PIP) binding proteins and function as repressors of Akt. They have tumor suppressive functions, mainly through Akt inhibition. Several reports suggest that PHLDA1 also has a tumor suppressive function; however, the precise molecular functions of PHLDA1 remain to be elucidated. Through a comprehensive screen for p53 target genes, we identified PHLDA1 as a novel p53 target, and we show that PHLDA1 has the ability to repress Akt in a manner similar to that of PHLDA3 and PHLDA2. PHLDA1 has a so-called split PH domain in which the PH domain is divided into an N-terminal (ß sheets 1-3) and a C-terminal (ß sheets 4-7 and an α-helix) portions. We show that the PH domain of PHLDA1 is responsible for its localization to the plasma membrane and binding to phosphatidylinositol. We also show that the function of the PH domain is essential for Akt repression. In addition, PHLDA1 expression analysis suggests that PHLDA1 has a tumor suppressive function in breast and ovarian cancers.

Skewing of peritoneal resident macrophages toward M1-like is involved in enhancement of inflammatory responses induced by secondary necrotic neutrophils in aged mice.

Secondary necrotic cells, which are generated if apoptotic cells are incompletely cleared, induce severe inflammatory responses involving MIP-2 production and subsequent neutrophil infiltration. Recently, we showed that the phagocytic capacity of peritoneal resident macrophages from wild type (WT) aged mice as well as SMP30(-/-) mice fed a VC-limited diet as to secondary necrotic cells was reduced as compared with that in young mice, and that the inflammatory responses induced were stronger than those in young mice, presumably because of the delay in removal of secondary necrotic cells in aged mice. In this study, we investigated why MIP-2 production was increased in aged mice upon injection of secondary necrotic cells and why the phagocytic capacity of peritoneal resident macrophages from aged mice was reduced. When cocultured with secondary necrotic cells, the peritoneal resident macrophages from both types of aged mice significantly produced MIP-2 even in the absence of IFN-γ, whereas MIP-2 production by macrophages from WT young mice required IFN-γ. The peritoneal resident macrophages from both types of aged mice expressed CD40, a M1 macrophage marker, as in the case of M1 macrophages, which were obtained by treatment of macrophages from WT young mice with IFN-γ and LPS. Furthermore, M1 macrophages exhibited less phagocytic capacity as to secondary necrotic cells than non-treated macrophages. These results suggest that the phenotype of peritoneal resident macrophages is skewed toward M1-like in aged mice and that such skewing toward M1-like is involved in enhancement of inflammatory responses induced by secondary necrotic neutrophils in aged mice.

Suppression of MIP-2 or IL-8 production by annexins A1 and A4 during coculturing of macrophages with late apoptotic human peripheral blood neutrophils.

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils.

Cytokine secretion from human monocytes potentiated by P-selectin-mediated cell adhesion.

BACKGROUND/AIM: P-selectin is a carbohydrate-recognizing cell adhesion molecule expressed on activated platelets and endothelial cells. It plays a crucial role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. Cell adhesion mediated by P-selectin induces leukocyte activation, such as the generation of reactive oxygen species and the expression of blood coagulation factors. We assessed how P-selectin-mediated cell adhesion affects cytokine secretion from monocytes. METHODS: Human peripheral blood monocytes were cultured in a plate that had been coated with P-selectin purified from human platelets, and cytokines released in the culture supernatant from monocytes were determined by ELISA. RESULTS: The secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-12 and macrophage inflammatory protein-1ß increased 3- to 10-fold in response to P-selectin compared with unstimulated monocytes. We next examined the effects of cytokine treatment of monocytes on their susceptibility to P-selectin. The secretion of TNF-α from monocytes in response to P-selectin was increased when monocytes were preincubated with granulocyte/macrophage colony-stimulating factor, monocyte chemotactic protein-1 or interferon-γ (IFN-γ); IFN-γ was the most effective in potentiating TNF-α secretion from monocytes. CONCLUSION: These results suggest that the interaction of monocytes with P-selectin plays an important role not only in their trafficking but also in the regulation of cytokine production by these cells.

Regulation of the estrous cycle by neutrophils via opioid peptides.

We found previously that neutrophil-depleted mice exhibited significant blockading of both the regular estrous cycle and cyclic changes of steroid hormone levels. In this study, we aimed at elucidation of the underlying mechanism. To examine the possibility that an increase in bacteria in the vaginal vault of neutrophil-depleted mice causes blockading of the estrous cycle, we treated neutrophil-depleted mice with antibiotics but failed to restore the estrous cycle. We then examined another possibility that neutrophils regulate the estrous cycle via opioid peptides, because opioid peptides regulate steroidogenesis in theca and granulosa cells in the ovaries, and because neutrophils contain opioid peptides. In support of this possibility, naloxone, an opioid antagonist, blocked the estrous cycle and a µ opioid receptor agonist restored the estrous cycle in neutrophil-depleted mice. Pro-opiomelanocortin was immunohistochemically detected in peripheral blood neutrophils but not in ones that had infiltrated into the ovaries. i.v. injection of anti-MIP-2 polyclonal Ab caused blockading of the estrous cycle, whereas MIP-2 was detected in the ovaries, suggesting a role of MIP-2 in the regulation of the estrous cycle. Moreover, i.v. injection of MIP-2 decreased the pro-opiomelanocortin signal in peripheral blood neutrophils and caused blockading of the estrous cycle. Together, these results suggest that neutrophils maintain the estrous cycle via opioid peptides.

Interleukin-9 receptor gene is transcriptionally regulated by nucleolin in T-cell lymphoma cells.

Interleukin-9 (IL-9) is a multifunctional cytokine that not only has roles in immune and inflammatory responses but also is involved in growth-promoting and anti-apoptotic activities in multiple transformed cell lines, which suggests a potential role in tumorigenesis. Over-expression of the receptor of IL-9 (IL-9R) occurs in several types of human leukemias and in radiation-induced mouse T-cell lymphoma (TL). The molecular mechanism that regulates transcription of the IL-9R gene (Il9r) during leukemogenesis is, however, not well understood. Using a mouse TL cell line that has high expression of Il9r, we sought to dissect its promoter structure. Here we show that the active promoter for Il9r is located in the 5'-flanking AT-rich region. Chromatin immunoprecipitation showed the opening of chromatin structure of the promoter region coupled with nucleolin binding in vivo. Immunohistochemical analysis confirmed the increased localization of nucleolin in the nuclei of TL cells. These data indicate that increased expression of Il9r is associated with an increased binding of nucleolin, coupled with chromatin opening, to an AT-rich region in the 5'-flanking region of Il9r in TL cells.

The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice.

During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-α production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24 h. There were increases in the numbers of neutrophils and late apoptotic ones at 24 h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-α production at 36 h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12 h decreased the numbers of neutrophils and late apoptotic ones at 24 h, and thereafter TNF-α production at 36 h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-α production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses.

The importance of infiltrating neutrophils in SDF-1 production leading to regeneration of the thymus after whole-body X-irradiation.

Our previous study demonstrated that neutrophils transiently infiltrated into a site where apoptosis had been induced. However, the role of infiltrating neutrophils has not been fully elucidated. In this study, we examined their role in regeneration of the thymus after whole-body X-irradiation by focusing on SDF-1 production. After X-irradiation, the thymus became severely atrophied presumably due to phagocytosis of apoptotic thymocytes. At that time, a significant number of neutrophils were detected in the thymus. The thymus was then partially regenerated on day 7, whereas the level of SDF-1 in it was significantly increased on days 3 and 5. Depletion of neutrophils greatly impaired SDF-1 production and the thymus regeneration. Moreover, administration of a CXCR4 antagonist also greatly suppressed the thymus regeneration. Furthermore, coculturing of a stromal cell line with infiltrating neutrophils increased SDF-1 production. These results suggest that infiltrating neutrophils play an auxiliary role in regeneration of the thymus after whole-body X-irradiation through augmentation of SDF-1 production.

Apoptotic neutrophils and nitric oxide regulate cytokine production by IFN-γ-stimulated macrophages.

Early apoptotic neutrophils but not secondary necrotic ones down-regulate LPS-induced proinflammatory cytokine production of macrophages, thereby contributing to the resolution of inflammation. IFN-γ is also a well-known stimulant of macrophages, but how the apoptotic neutrophils affect IFN-γ-stimulated macrophages remains largely unexplored. Since IFN-γ induces the expression of inducible nitric oxide (NO) synthase, we examined the production of NO and various cytokines, including MIP-2, TNF-α, IL-12p40, IL-6, IL-10, and TGF-ß, by IFN-γ-stimulated murine macrophages, the effect of coculturing the macrophages with early apoptotic or secondary necrotic neutrophils, and the regulatory role of NO in such cocultures. IFN-γ induced significant production of NO, IL-12p40, and IL-6 by macrophages, but not other cytokines. Early apoptotic neutrophils but not secondary necrotic ones promoted NO production, whereas secondary necrotic ones and their supernatants promoted TNF-α production. In contrast, both early apoptotic and secondary necrotic neutrophils suppressed IL-12p40 and IL-6 production. Furthermore, macrophages from inducible NO synthase-deficient mice produced significantly higher levels of MIP-2 than those from wild-type mice. Consistent with this, treatment of macrophages with l-NAME, an NO synthase inhibitor, also induced the production of a large amount of MIP-2. In conclusion, this study suggests that early apoptotic neutrophils are critical in the resolution of inflammation, but that secondary necrotic neutrophils may not cause an inflammatory response. Apoptotic neutrophils, however, appear not to modulate cytokine production via NO.

RSV infection-elicited high MMP-12-producing macrophages exacerbate allergic airway inflammation with neutrophil infiltration.

Respiratory syncytial virus (RSV) infection often exacerbates bronchial asthma, but there is no licensed RSV vaccine or specific treatments. Here we show that RSV-induced alveolar macrophages, which produce high levels of matrix metalloproteinase-12 (MMP-12), exacerbate allergic airway inflammation with increased neutrophil infiltration. When mice subjected to allergic airway inflammation via exposure to the house dust mite antigen (HDM) were infected with RSV (HDM/RSV), MMP-12 expression, viral load, neutrophil infiltration, and airway hyperresponsiveness (AHR) were increased compared to those in the HDM and RSV groups. These exacerbations in the HDM/RSV group were attenuated in MMP-12-deficient mice and mice treated with MMP408, a selective MMP-12 inhibitor, but not in mice treated with dexamethasone. Finally, M2-like macrophages produced MMP-12, and its production was promoted by increase of IFN-ß-induced IL-4 receptor expression with RSV infection. Thus, targeting MMP-12 represents a potentially novel therapeutic strategy for the exacerbation of asthma.

Respiratory syncytial virus infection exacerbates pneumococcal pneumonia via Gas6/Axl-mediated macrophage polarization.

Patients with respiratory syncytial virus (RSV) infection exhibit enhanced susceptibility to subsequent pneumococcal infections. However, the underlying mechanisms involved in this increased susceptibility remain unclear. Here, we identified potentially novel cellular and molecular cascades triggered by RSV infection to exacerbate secondary pneumococcal pneumonia. RSV infection stimulated the local production of growth arrest-specific 6 (Gas6). The Gas6 receptor Axl was crucial for attenuating pneumococcal immunity in that the Gas6/Axl blockade fully restored antibacterial immunity. Mechanistically, Gas6/Axl interaction regulated the conversion of alveolar macrophages from an antibacterial phenotype to an M2-like phenotype that did not exhibit antibacterial activity, and the attenuation of caspase-1 activation and IL-18 production in response to pneumococcal infection. The attenuated IL-18 production failed to drive both NK cell-mediated IFN-γ production and local NO and TNF-α production, which impair the control of bacterial infection. Hence, the RSV-mediated Gas6/Axl activity attenuates the macrophage-mediated protection against pneumococcal infection. The Gas6/Axl axis could be a potentially novel therapeutic target for RSV-associated secondary bacterial infection.

Regulation of the estrous cycle by neutrophil infiltration into the vagina.

During metestrus of the estrous cycle, a number of neutrophils infiltrate into the vaginal vault, presumably due to a neutrophil-specific chemokine, MIP-2, in mice. The physiological role of the infiltrating neutrophils, however, remains largely obscure. In this study we examined the effects of neutrophil depletion on the estrous cycle and steroid hormone levels. When mice were treated with an anti-Gr-1 mAb, they became neutropenic, as assessed as to the number of neutrophils in the peripheral blood. The estrous cycle of such mice was specifically blocked at diestrus irrespective of the phase at which the anti-Gr-1 mAb was administered. The blockade was reversible, because restoration of neutrophils to a normal level caused a restart of the cycle. Immunohistochemical analyses revealed that neutrophils were present mainly on the luminal surface and in the lumen at metestrus and to a lesser extent at diestrus but scarcely in the uterine cervix at any phase, and that the anti-Gr-1 mAb depleted neutrophils but not eosinophils in the vagina. The treatment with the anti-Gr-1 mAb significantly affected the serum 17beta-estradiol and progesterone levels at diestrus after the estrous cycle was blocked. Together, these results suggest that neutrophil infiltration into the vagina is critical in maintaining the estrous cycle through control of steroid hormone levels.

Vitamin C deficiency causes muscle atrophy and a deterioration in physical performance.

L-Ascorbic acid (AsA) is a water-soluble antioxidant. We examined the effect of AsA deficiency on skeletal muscle using senescence marker protein-30 (SMP30)-knockout (KO) mice that are defective in AsA biosynthesis, which makes this mouse model similar to humans, to clarify the function of AsA in skeletal muscle. Eight-week-old female SMP30-KO mice were divided into the following two groups: an AsA-sufficient group [AsA(+)] that was administered 1.5 g/L AsA and an AsA-deficient group [AsA(-)] that was administered tap (AsA-free) water. At 4 weeks, the AsA content in the gastrocnemius muscle of AsA(-) mice was 0.7% compared to that in the gastrocnemius muscle of AsA(+) mice. Significantly lower weights of all muscles were observed in AsA(-) mice than those in AsA(+) mice at 12 and 16 weeks. The cross-sectional area of the soleus was significantly smaller in AsA(-) mice at 16 weeks than that in AsA(+) mice. The physical performance of AsA(-) mice was significantly less than that of AsA(+) mice at 12 weeks. Following AsA deficiency for 12 weeks, the expression of ubiquitin ligases, such as atrogin1/muscle atrophy F-box (MAFbx) and muscle RING-finger protein 1 (MuRF1), was upregulated. Furthermore, all detected effects of AsA deficiency on muscles of the AsA(-) group at 12 weeks were restored following AsA supplementation for 12 weeks. Thus, longer-term AsA deficiency is associated with muscle wasting, that this can be reversed by restoring AsA levels.

Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.

Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naïve T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1(-) and Gr-1(+) ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.

Transient infiltration of neutrophils into the thymus following whole-body X-ray irradiation in IL-10 knockout mice.

IL-10 is known to suppress the inflammatory responses in a variety of experimental models. Because we previously found that whole-body X-irradiation causes massive apoptosis in the thymus and transient infiltration of neutrophils, in this study, we examined whether or not IL-10 is involved in the regulation of neutrophil infiltration upon whole-body X-ray irradiation using IL-10 knockout mice. Although IL-10 was induced in the thymus on whole-body X-ray irradiation, apoptosis of thymocytes, neutrophil infiltration, and MIP-2 and KC production in the thymus were not affected by an IL-10 deficiency. Coculturing of bone marrow-derived macrophages with late apoptotic cells caused MIP-2 production, which was also not affected by an IL-10 deficiency. These results suggest the uniqueness of the inflammatory response induced by whole-body X-ray irradiation, which does not seem to be regulated by IL-10.

Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrow-derived macrophages upon coculturing with late apoptotic cells.

Recently, we found that resident peritoneal macrophages produce MIP-2, one of the major chemokines for neutrophils, upon coculturing with late apoptotic cells, and that intraperitoneal injection of late apoptotic cells into the peritoneal cavity causes neutrophil infiltration via MIP-2. It is not known, however, whether or not macrophages are heterogeneous in such MIP-2 production. In this study, we examined changes in the surface phenotype during the differentiation of bone marrow cells into macrophages due to M-CSF and GM-CSF, and then examined the production of cytokines, namely IL-12 p40, MIP-2, IL-10, and TGF-beta, following phagocytosis of late apoptotic cells with these macrophages or LPS stimulation of these macrophages. GM-CSF and M-CSF induced macrophage populations with distinct but overlapping cell surface phenotype. Although these macrophages phagocytosed late apoptotic cells to a similar extent, they produced either IL-12 p40 or IL-10, whereas they produced MIP-2 to a similar extent after the coculture, raising the possibility that late apoptotic cells may induce neutrophil infiltration in any organs, such as the liver and lungs, where the macrophages are differentiated by either M-CSF or GM-CSF, respectively.

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis.

TNF-alpha, IFN-gamma, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-alpha production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-gamma, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-gamma, which was slightly suppressed at 12h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-alpha in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.

Infiltrating neutrophils induce allospecific CTL in response to immunization with apoptotic cells via MCP-1 production.

Our previous studies demonstrated that i.p. injection of late apoptotic P388 cells caused phagocytosis by macrophages and transient infiltration of neutrophils into the peritoneal cavity. As neutrophils are known to function as effectors as well as regulators in the immune response, we examined the roles of infiltrating neutrophils in alloantigen-specific CTL induction after immunization with late apoptotic P388 cells. The CTL induction and infiltration of CD8(+) T cells into the peritoneal cavity were inhibited by depletion of neutrophils by anti-Gr-1 mAb or inhibition of neutrophil infiltration by anti-MIP-2 antibody, suggesting that neutrophils are involved in CD8(+) T cell infiltration into the peritoneal cavity. It is known that MIP-1alpha, MIP-1beta, and MCP-1 are capable of attracting CD8(+) T cells and that they are produced by neutrophils. These chemokines were detected in the peritoneal cavity, and among them, MCP-1 production was reduced remarkably by suppression of neutrophil infiltration. Moreover, infiltration of CD8(+) T cells into the peritoneal cavity as well as CTL activity was clearly reduced by administering anti-MCP-1 antibody i.p. Furthermore, the CTL induction and infiltration of CD8(+) T cells in neutrophil-depleted mice were restored significantly by administering recombinant murine MCP-1 into the peritoneal cavity. These results indicate that MCP-1 appears to link infiltration of neutrophils with CTL induction.

Redistribution of P-selectin glycoprotein ligand-1 (PSGL-1) in chemokine-treated neutrophils: a role of lipid microdomains.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like cell adhesion molecule expressed on leukocyte plasma membranes and involved in platelet-leukocyte and endothelium-leukocyte interactions. The treatment of neutrophils with a low concentration of IL-8 induced the redistribution of PSGL-1 to one end of the cell to form a cap-like structure. We investigated the role of lipid microdomains in the redistribution of PSGL-1 and its effect on the adhesive characteristics of IL-8-treated neutrophils. The redistribution of PSGL-1 induced by IL-8 was inhibited by cholesterol-perturbing agents such as methyl-beta-cyclodextrin and filipin. Sucrose density gradient centrifugation analysis revealed that PSGL-1 was enriched in a low-density fraction together with the GM1 ganglioside after solubilization of the cell membranes with a nonionic detergent, Brij 58. However, when Triton X-100 was used for the solubilization, PSGL-1 was no longer recovered in the low-density fraction, although GM1 ganglioside remained in the low-density fraction. Furthermore, immunofluorescence microscopic observation demonstrated that the localization of PSGL-1 differed from that of GM1 ganglioside, suggesting that PSGL-1 is associated with a microdomain distinct from that containing the GM1 ganglioside. Treatment of neutrophils with IL-8 increased the formation of microaggregates composed of neutrophils and activated platelets, and this treatment also enhanced reactive oxygen species production in neutrophils induced by the cross-linking of PSGL-1 with antibodies. These results suggest that the association of PSGL-1 with lipid microdomains is essential for its redistribution induced by IL-8 stimulation and that the redistribution modulates neutrophil functions mediated by interactions with P-selectin.