The Impact of Integrin ß2 on Granulocyte/Macrophage Progenitor Proliferation.

Previously, we reported that although the HSPC frequency in bone marrow cells (BMC) was comparable between ß2-/- and ß2+/+ mice, transplantation of ß2-/- BMC into lethally irradiated CD45.1 recipient resulted in more myeloid cell production than ß2+/+ BMC. The objective of this study is to address if integrin ß2 deficiency skews granulocyte/macrophage progenitor (GMP) proliferation. FACS analysis demonstrated that GMP frequency and cell number were higher and megakaryocyte/erythrocyte progenitor frequency and cell number were lower in ß2-/- mice than ß2+/+ mice. However, the common myeloid progenitors (CMP) frequency and cell number were similar between the two groups. The increased GMP number was due to GMP proliferation as evidenced by the percentage of BrdU-incorporating GMP. Whole genome transcriptome analysis identified increased FcεRIα expression in ß2-/- CMP compared to ß2+/+ CMP. FcεRIα expression on ß2-/- GMP was detected increased in ß2-/- mice by qRT-PCR and FACS. Although transplantation of FcεRIαhi GMP or FcεRIαlo GMP into lethally irradiated CD45.1 recipient resulted in comparable myeloid cell production, transplantation of ß2 deficient FcεRIαhi GMP generated more myeloid cells than ß2+/+ FcεRIαhi GMP. GATA2 expression was increased in ß2-/- GMP. Using a luciferase reporter assay, we demonstrated that mutation of the GATA2 binding site in the FcεRIα promoter region diminished FcεRIα transcription. In vitro, the addition of IgE, the ligand of FcεRIα, promoted GMP expansion, which was abrogated by inhibition of JNK phosphorylation. Integrin ß2 deficiency promoted GMP proliferation and myeloid cell production, which was mediated via FcεRIα/IgE-induced JNK phosphorylation in GMP. Stem Cells 2019;37:430-440.

S1-Guidelines on UV phototherapy and photochemotherapy.

Known in part since antiquity, the salutary effects of sunlight again garnered increasing attention in the second half of the 19(th) century. The development of a device for ultraviolet irradiation of cutaneous tuberculosis by Finnsen at the onset of the twentieth century truly marked the beginning of modern phototherapy. In dermatology, treatment methods almost exclusively use wavelengths below the visible light range (ultraviolet light). Since the early 1970s, increasingly powerful artificial light sources have become available for UVB and UVA therapy as well as the combination of UVA and photosensitizers (photochemotherapy). High structural and procedural quality standards are an essential prerequisite for the implementation of effective as well as safe phototherapy. The following guidelines outline the current consensus of leading experts in the field of phototherapy with respect to indications, contraindications, and side effects of various treatment options available. Particular focus is also on adequate UV doses at the beginning and over the further course of treatment as well as on management of side effects.

Hematopoietic stem/progenitor cells directly contribute to arteriosclerotic progression via integrin ß2.

Recent studies described the association between hematopoietic stem/progenitor cell (HSPC) expansion in the bone marrow (BM), leukocytosis in the peripheral blood, and accelerated atherosclerosis. We hypothesized that circulating HSPC may home to inflamed vessels, where they might contribute to inflammation and neointima formation. We demonstrated that Lin(-) Sca-1(+) cKit(+) (LSK cells) in BM and peripheral blood of LDLr(-/-) mice on high fat diet expressed significantly more integrin ß2 , which was responsible for LSK cell adhesion and migration toward ICAM-1 in vitro, and homing to injured arteries in vivo, all of which were blocked with an anti-CD18 blocking antibody. When homed LSK cells were isolated from ligated artery and injected to irradiated recipients, they resulted in BM reconstitution. Injection of CD18(+/+) LSK cells to immunodeficient Balb/C Rag2(-) É£C(-/-) recipients resulted in more severe inflammation and reinforced neointima formation in the ligated carotid artery, compared to mice injected with PBS and CD18(-/-) LSK cells. Hypercholesterolemia stimulated ERK phosphorylation (pERK) in LSK cells of LDLr(-/-) mice in vivo. Blockade of pERK reduced ARF1 expression, leading to decreased integrin ß2 function on HSPC. In addition, integrin ß2 function could be regulated via ERK-independent LRP1 pathway. Integrin ß2 expression on HSPC is regulated by hypercholesterolemia, specifically LDL, in pERK-dependent and -independent manners, leading to increased homing and localization of HSPC to injured arteries, which is highly correlated with arteriosclerosis.

ß2 integrin mediates hantavirus-induced release of neutrophil extracellular traps.

Rodent-borne hantaviruses are emerging human pathogens that cause severe human disease. The underlying mechanisms are not well understood, as hantaviruses replicate in endothelial and epithelial cells without causing any cytopathic effect. We demonstrate that hantaviruses strongly stimulated neutrophils to release neutrophil extracellular traps (NETs). Hantavirus infection induced high systemic levels of circulating NETs in patients and this systemic NET overflow was accompanied by production of autoantibodies to nuclear antigens. Analysis of the responsible mechanism using neutrophils from ß2 null mice identified ß2 integrin receptors as a master switch for NET induction. Further experiments suggested that ß2 integrin receptors such as complement receptor 3 (CR3) and 4 (CR4) may act as novel hantavirus entry receptors. Using adenoviruses, we confirmed that viral interaction with ß2 integrin induced strong NET formation. Collectively, ß2 integrin-mediated systemic NET overflow is a novel viral mechanism of immunopathology that may be responsible for characteristic aspects of hantavirus-associated disease such as kidney and lung damage.

An unrestrained proinflammatory M1 macrophage population induced by iron impairs wound healing in humans and mice.

Uncontrolled macrophage activation is now considered to be a critical event in the pathogenesis of chronic inflammatory diseases such as atherosclerosis, multiple sclerosis, and chronic venous leg ulcers. However, it is still unclear which environmental cues induce persistent activation of macrophages in vivo and how macrophage-derived effector molecules maintain chronic inflammation and affect resident fibroblasts essential for tissue homeostasis and repair. We used a complementary approach studying human subjects with chronic venous leg ulcers, a model disease for macrophage-driven chronic inflammation, while establishing a mouse model closely reflecting its pathogenesis. Here, we have shown that iron overloading of macrophages--as was found to occur in human chronic venous leg ulcers and the mouse model--induced a macrophage population in situ with an unrestrained proinflammatory M1 activation state. Via enhanced TNF-α and hydroxyl radical release, this macrophage population perpetuated inflammation and induced a p16(INK4a)-dependent senescence program in resident fibroblasts, eventually leading to impaired wound healing. This study provides insight into the role of what we believe to be a previously undescribed iron-induced macrophage population in vivo. Targeting this population may hold promise for the development of novel therapies for chronic inflammatory diseases such as chronic venous leg ulcers.

Extracellular adherence protein of Staphylococcus aureus suppresses disease by inhibiting T-cell recruitment in a mouse model of psoriasis.

Psoriasis is a T-cell-mediated inflammatory disease. Previous studies focused on lymphocyte function-associated antigen 1 (LFA-1)-expressing T cells as a molecular target for therapeutic intervention. By contrast, information on therapeutic effects and the underlying mechanism of blocking the LFA-1 counter receptor, ICAM-1 is scarce. Here, we used the CD18 (beta2-integrin) hypomorphic (CD18hypo) mouse model of psoriasis to investigate the therapeutic role of extracellular adherence protein (Eap) of Staphylococcus aureus, which exerts antiinflammatory activities by interacting with the ICAM-1 function. We show that ICAM-1 is predominantly upregulated on endothelial cells in lesional skin of CD18hypo mice. In vitro Eap was found to disrupt cell-cell contacts between T cells and dendritic cells, and inhibit T-cell proliferation. By contrast, in vivo Eap rather blocked transmigration of T cells from vessels to inflamed skin of CD18hypo mice, but did not inhibit their proliferation and activation. Most importantly, Eap successfully suppressed the disease by blocking T-cell extravasation into the inflamed skin. Together, these data indicate that interaction between LFA-1 and ICAM-1 is causally involved in the pathogenesis of psoriasiform skin inflammation, and targeting ICAM-1 to selectively block T-cell extravasation by Eap without immune suppression may represent a potential therapeutic strategy for psoriasis.

Key role of macrophages in the pathogenesis of CD18 hypomorphic murine model of psoriasis.

Psoriasis is a chronic skin disorder of unsolved pathogenesis affecting skin in 2-3% of the general population. Research into the pathogenesis of psoriasis has profited from suitable animal models. Previously, we reported on the CD18 hypomorphic (CD18(hypo)) PL/J mouse model clinically resembling human psoriasis, which is characterized by reduced expression of the common chain of beta(2)-integrins (CD11/CD18) to only 2-16% of wild-type levels. Aside from common clinical and pathophysiological features shared with human psoriasis, the psoriasiform skin disease in CD18(hypo) PL/J mice also depends on the presence of CD4(+) T-cells. This review focuses on the role of activated macrophages in the pathogenesis of CD18(hypo) T-cell-mediated mouse model of psoriasis, and extends our understanding in unrestrained pathogenic T-cells whose activation may be crucial for the recruitment and activation of macrophages within skin. The findings in the CD18(hypo) PL/J model are discussed in the context of current literatures of human and other autoimmune disorders.

Wound healing defect of Vav3-/- mice due to impaired {beta}2-integrin-dependent macrophage phagocytosis of apoptotic neutrophils.

Vav proteins are guanine-nucleotide exchange factors implicated in leukocyte functions by relaying signals from immune response receptors and integrins to Rho-GTPases. We here provide first evidence for a role of Vav3 for beta(2)-integrins-mediated macrophage functions during wound healing. Vav3(-/-) and Vav1(-/-)/Vav3(-/-) mice revealed significantly delayed healing of full-thickness excisional wounds. Furthermore, Vav3(-/-) bone marrow chimeras showed an identical healing defect, suggesting that Vav3 deficiency in leukocytes, but not in other cells, is causal for the impaired wound healing. Vav3 was required for the phagocytotic cup formation preceding macrophage phagocytosis of apoptotic neutrophils. Immunoprecipitation and confocal microscopy revealed Vav3 activation and colocalization with beta(2)-integrins at the macrophage membrane upon adhesion to ICAM-1. Moreover, local injection of Vav3(-/-) or beta(2)-integrin(CD18)(-/-) macrophages into wound margins failed to restore the healing defect of Vav3(-/-) mice, suggesting Vav3 to control the beta(2)-integrin-dependent formation of a functional phagocytic synapse. Impaired phagocytosis of apoptotic neutrophils by Vav3(-/-) macrophages was causal for their reduced release of active transforming growth factor (TGF)-beta(1), for decreased myofibroblasts differentiation and myofibroblast-driven wound contraction. TGF-beta(1) deficiency in Vav3(-/-) macrophages was causally responsible for the healing defect, as local injection of either Vav3-competent macrophages or recombinant TGF-beta(1) into wounds of Vav3(-/-) mice fully rescued the delayed wound healing.

A 9-centimorgan interval of chromosome 10 controls the T cell-dependent psoriasiform skin disease and arthritis in a murine psoriasis model.

Psoriasis is a complex genetic disease of unresolved pathogenesis with both heritable and environmental factors contributing to onset and severity. In addition to a disfiguring skin inflammation, approximately 10-40% of psoriasis patients suffer from destructive joint involvement. Previously, we reported that the CD18 hypomorphic PL/J mouse carrying a mutation resulting in reduced expression of the common chain of beta(2) integrins (CD11/CD18) spontaneously develops a skin disease that closely resembles human psoriasis. In contrast, the same mutation on C57BL/6J background did not demonstrate this phenotype. By a genome-wide linkage analysis, two major loci were identified as contributing to the development of psoriasiform dermatitis under the condition of low CD18 expression. Using a congenic approach, we now demonstrate that the introduction of a 9-centimorgan fragment of chromosome 10 derived from the PL/J strain into the disease-resistant CD18 hypomorphic C57BL/6J was promoting the development of psoriasiform skin disease and notably also arthritis. We therefore designated this locus psoriasiform skin disease-associated locus 1 (PSD1). High numbers of CD4(+) T cells and TNF-alpha producing macrophages were detected both in inflamed skin and joints in these congenic mice, with a complete resolution upon TNF-alpha inhibitor therapy or depletion of CD4(+) T cells. For the first time, we have identified a distinct genetic element that contributes to the T cell-dependent development of both psoriasiform skin disease and associated arthritis. This congenic model will be suitable to further investigations of genetic and molecular pathways that cause psoriasiform dermatitis and arthritis, and it may also be relevant for other autoimmune diseases.

Differential molecular and anatomical basis for B cell migration into the peritoneal cavity and omental milky spots.

The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that alpha(4)beta(7) integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, alpha(4)beta(1) integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.

Beta2 integrins separate graft-versus-host disease and graft-versus-leukemia effects.

Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation. Migration of donor-derived T cells into GVHD target organs plays an essential role in the development of GVHD. beta2 integrins are critically important for leukocyte extravasation through vascular endothelia and for T-cell activation. We asked whether CD18-deficient T cells would induce less GVHD while sparing the graft-versus-leukemia (GVL) effect. In murine allogeneic bone marrow transplantation models, we found that recipients of CD18-/- donor T cells had significantly less GVHD morbidity and mortality compared with recipients of wild-type (WT) donor T cells. Analysis of alloreactivity showed that CD18-/- and WT T cells had comparable activation, expansion, and cytokine production in vivo. Reduced GVHD was associated with a significant decrease in donor T-cell infiltration of recipient intestine and with an overall decrease in pathologic scores in intestine and liver. Finally, we found that the in vivo GVL effect of CD18-/- donor T cells was largely preserved, because mortality of the recipients who received transplants of CD18-/- T cells plus tumor cells was greatly delayed or prevented. Our data suggest that strategies to target beta2 integrin have clinical potential to alleviate or prevent GVHD while sparing GVL activity.

RhoH is important for positive thymocyte selection and T-cell receptor signaling.

RhoH is a small GTPase expressed only in the hematopoietic system. With the use of mice with targeted disruption of the RhoH gene, we demonstrated that RhoH is crucial for thymocyte maturation during DN3 to DN4 transition and during positive selection. Furthermore, the differentiation and expansion of DN3 and DN4 thymocytes in vitro were severely impaired. These defects corresponded to defective TCR signaling. Although RhoH is not required for TCR-induced activation of ZAP70 and ZAP70-mediated activation of p38, it is crucial for the tyrosine phosphorylation of LAT, PLCgamma1, and Vav1 and for the activation of Erk and calcium influx. These data suggest that RhoH is important for pre-TCR and TCR signaling because it allows the efficient interaction of ZAP70 with the LAT signalosome, thus regulating thymocyte development.

Pathogenic role for skin macrophages in a mouse model of keratinocyte-induced psoriasis-like skin inflammation.

Psoriasis is a common skin disease, the pathogenesis of which has not yet been resolved. In mice, epidermis-specific deletion of inhibitor of NF-kappaB (IkappaB) kinase 2 (IKK2) results in a skin phenotype that mimics human psoriasis in several aspects. Like psoriasis, this skin disease shows pronounced improvement when mice are treated with a TNF-neutralizing agent. We have found previously that this phenotype does not depend on the presence of alphabeta T lymphocytes. In order to evaluate contributions of other immune cell populations to the skin disease, we selectively eliminated macrophages and granulocytes from the skin of mice with epidermis-specific deletion of IKK2 (K14-Cre-IKK2fl/fl mice). Elimination of skin macrophages by subcutaneous injection of clodronate liposomes was accompanied by inhibition of granulocyte migration into the skin and resulted in a dramatic attenuation of psoriasis-like skin changes. The hyperproliferative, inflammatory skin disease in K14-Cre-IKK2fl/fl mice was a direct consequence of the presence of macrophages in the skin, as targeted deletion of CD18, which prevented accumulation of granulocytes but not macrophages, did not lead to major changes in the phenotype. Targeted deletion of the receptor for IFN-gamma revealed that the pathogenesis of the skin disease does not depend on classical IFN-gamma-mediated macrophage activation. Our results demonstrate that in mice epidermal keratinocytes can initiate a hyperproliferative, inflammatory, IFN-gamma-independent, psoriasis-like skin disease whose development requires essential contributions from skin macrophages but not from granulocytes or alphabeta T lymphocytes.

Terminal B cell differentiation is skewed by deregulated interleukin-6 secretion in beta2 integrin-deficient mice.

Absence of the common beta chain (CD18) of beta(2) integrins leads to leukocyte-adhesion deficiency type-1 (LAD1) in humans. Mice with a CD18 null mutation suffer from recurrent bacterial infections, impaired wound healing, and skin ulcers, closely resembling human LAD1. Previous findings in CD18(-/-) mice demonstrated a skewed terminal B cell differentiation with plasmacytosis and elevated serum immunoglobulin G (IgG). As interleukin-6 (IL-6) is a potent enhancer of plasma cell formation and Ig secretion, we assessed IL-6 serum levels of CD18(-/-) and wild-type (WT) mice kept under a conventional or barrier facility or specific pathogen-free (SPF) conditions. We detected an up to 20-fold increase in IL-6 in serum of CD18(-/-) mice compared with WT controls when kept under conventional or barrier facility conditions, respectively. Under SPF conditions, no significant differences in terms of IL-6 serum levels were found between CD18(-/-) and WT mice. However, histological alterations of secondary lymphoid tissues, plasmacytosis, abnormal plasmacytoid cells (Mott cells), and hypergammaglobulinemia persisted. To further analyze the role of IL-6 in these pathological alterations, we established a CD18(-/-) IL-6(-/-) double-deficient mouse mutant. In these mice, serum IgG levels were normal, and the altered plasma cell phenotype, including Mott cells, was no longer detectable. The CD18(-/-) IL-6(-/-) double-deficient mouse model thus demonstrated that IL-6 is responsible for parts of the phenotype seen in the CD18(-/-) mouse mutants. It may be of interest to examine human leukocyte-adhesion deficiency type-1 patients closer and search for pathological changes possibly induced via overproduction of IL-6.

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.

Cutting edge: egress of newly generated plasma cells from peripheral lymph nodes depends on beta 2 integrin.

During humoral immune responses, naive B cells differentiate into Ab-secreting plasma cells within secondary lymphoid organs. Differentiating plasma cells egress from their sites of generation and redistribute to other tissues, predominantly the bone marrow and mucosal tissues. In this study, we demonstrate that within peripheral lymph nodes newly generated plasma cells localize to medullary cords which express the beta(2) integrin ligand ICAM-1. In beta(2) integrin-deficient mice plasma cells accumulate inside the lymph nodes, resulting in severely reduced plasma cell numbers in the bone marrow. Since plasma cells isolated from beta(2) integrin-deficient animals migrate efficiently into the bone marrow when transferred i.v., our findings provide profound evidence that beta(2) integrins are required for the egress of plasma cells from peripheral lymph nodes.

Role of beta2-integrins for homing and neovascularization capacity of endothelial progenitor cells.

The mechanisms of homing of endothelial progenitor cells (EPCs) to sites of ischemia are unclear. Here, we demonstrate that ex vivo-expanded EPCs as well as murine hematopoietic Sca-1+/Lin- progenitor cells express beta2-integrins, which mediate the adhesion of EPCs to endothelial cell monolayers and their chemokine-induced transendothelial migration in vitro. In a murine model of hind limb ischemia, Sca-1+/Lin- hematopoietic progenitor cells from beta2-integrin-deficient mice are less capable of homing to sites of ischemia and of improving neovascularization. Preactivation of the beta2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo. These results provide evidence for a novel function of beta2-integrins in postnatal vasculogenesis.

Overexpression of phospholipid-hydroperoxide glutathione peroxidase in human dermal fibroblasts abrogates UVA irradiation-induced expression of interstitial collagenase/matrix metalloproteinase-1 by suppression of phosphatidylcholine hydroperoxide-mediated NFkappaB activation and interleukin-6 release.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) exhibits high specific activity in reducing phosphatidylcholine hydroperoxides (PCOOHs) and thus may play a central role in protecting the skin against UV irradiation-triggered detrimental long term effects like cancer formation and premature skin aging. Here we addressed the role of PHGPx in the protection against UV irradiation-induced expression of matrix metalloproteinase-1 (MMP-1). For this purpose, we created human dermal fibroblast cell lines overexpressing human PHGPx. Overexpression led to a significant increase in PHGPx activity. In contrast to a maximal 4.5-fold induction of specific MMP-1 mRNA levels in vector-transfected cells at 24 h after UVA irradiation, no MMP-1 induction occurred at any studied time point after UVA treatment of PHGPx-overexpressing fibroblasts. As interleukin-6 (IL-6) was earlier shown to mediate the UVA induction of MMP-1, we studied whether PHGPx overexpression might interfere with the NFkappaB-mediated IL-6 induction and downstream signaling. Using transient transfections of IL-6 promoter constructs containing NFkappaB binding sites, we observed a high induction of the reporter gene luciferase in vector-transfected control cells and a significantly lower induction in PHGPx-overexpressing fibroblasts following UVA irradiation. Consistently both UVA irradiation and treatment of fibroblasts with PCOOHs led to phosphorylation and nuclear translocation of the p65 subunit, whereas cells overexpressing PHGPx exhibited impaired NFkappaB activation, p65 phosphorylation, and nuclear translocation. In line with this, the PHGPx-overexpressing fibroblasts showed a reduced constitutive and UVA irradiation-induced IL-6 release. After incubating PHGPx-overexpressing cells with PCOOHs a reduced induction of IL-6 was observed. This together with the suppression of UVA irradiation-induced IL-6 release in the presence of Trolox, a chain breaker of PCOOH-initiated lipid peroxidation, indicates that UVA irradiation-induced PCOOHs and subsequent lipid peroxides initiate the NFkappaB-mediated induction of IL-6, which mediates the induction of MMP-1. Our finding is particularly relevant in light of the already available small molecule mimetics of PHGPx.

A role for apoptosis in the control of neutrophil homeostasis in the circulation: insights from CD18-deficient mice.

The control of neutrophil turnover in the circulation is a key event in homeostasis and inflammation. Using CD18- deficient (CD18(-/-)) mice that show a 19-fold increase of blood neutrophil counts when compared with wild-type animals (CD18(+/+)), we found that apoptosis of peripheral neutrophils was significantly reduced from 27.4% in the wild-type to 4.8% in CD18(-/-) mice within 4 hours after isolation as measured by analysis of DNA content. This was confirmed by detecting CD16 expression, nuclear morphology, and internucleosomal DNA degradation. In contrast, no difference in apoptosis was observed in neutrophils derived from the bone marrow. Neutrophilia and delayed neutrophil apoptosis were also present in CD18(-/-)/interleukin 6 (IL-6(-/-)) double knockout mice. Moreover, plasma of CD18(-/-) mice was not able to delay apoptosis of CD18(+/+) neutrophils and plasma of CD18(+/+) mice did not augment apoptosis of CD18(-/-) neutrophils. However, CD18(-/-) neutrophils revealed an up-regulation of the antiapoptotic gene bcl-X(l) and a down-regulation of the proapoptotic gene bax-alpha compared with CD18(+/+) neutrophils suggesting that this delayed apoptosis. Accordingly, down-regulation of Bax-alpha using antisense technique delayed apoptosis and prolonged neutrophil survival. The replacement of the hematopoietic system of CD18(+/+) mice by a 1:1 mixture of CD18(+/+) and CD18(-/-) hematopoietic cells abolished the delay of apoptosis in peripheral CD18(-/-) neutrophils and prevented neutrophilia. Altogether, this suggests that a delay of neutrophil apoptosis in CD18(-/-) mice causes an alteration of neutrophil homeostasis, which may induce the massive increase of peripheral neutrophil counts. Thus, apoptosis seems to be critically involved in the control of neutrophil turnover in the circulation.