Neutrophil-depleted Mice Research Articles

Neutrophils Sequestered in the Liver Suppress the Proinflammatory Response of Kupffer Cells to Systemic Bacterial Infection

The liver plays a major role in clearing bacteria from the bloodstream. Rapid clearance is primarily the function of fixed tissue macrophages (Kupffer cells) that line the hepatic sinusoids. Although Kupffer cells play a critical role in blood clearance, the actual elimination of the bulk of bacteria taken up by the liver depends upon the accumulation of bactericidal neutrophils. Subsequent experiments demonstrating neutrophils inside Kupffer cells derived from infected animals prompted our speculation that neutrophils modulate the proinflammatory response of Kupffer cells to bacteria cleared from the bloodstream. Indeed, we report here that neutrophils accumulated in the liver sinusoids suppress cytokine and chemokine mRNA expression and protein production by Kupffer cells. Using listeriosis in mice as an experimental model, we found that IL-1beta, IL-6, IL-10, IL-12, TNF-alpha, MIP-1alpha, keratinocyte-derived chemokine, and MCP-1 mRNA levels were >or=10-fold more in the livers of Listeria-infected, relative to noninfected control, mice at 0.5-2 h after i.v. infection. Most message levels were sharply diminished thereafter, correlating inversely with increased neutrophil sequestration. Relative to intact animals, mice rendered neutrophil deficient exhibited marked increases in cytokine/chemokine mRNA expression and protein production in the liver subsequent to infection. Moreover, purified Kupffer cells derived from infected, neutrophil-depleted mice produced significantly more IL-6, IL-10, IL-12, TNF-alpha, keratinocyte-derived chemokine, and MCP-1 in culture. These findings document the critical role of neutrophils in moderating the proinflammatory response of Kupffer cells to bacteria taken up by the liver.

Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators

Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1alpha (Ccl3/MIP-1alpha) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1alpha induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B(4) (LTB(4)) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB(4), but not Ccl2/JE/MCP-1 and Ccl3/MIP-1alpha, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/MCP-1- and Ccl3/MIP-1alpha-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice. Ccl2/JE/MCP-1 and Ccl3/MIP-1alpha mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage.

The 2008 Dolph Adams Award and the State of the Journal of Leukocyte Biology

The 2008 Dolph Adams Award and the State of the <i>Journal of Leukocyte Biology</i>

Leukotriene B4 Induces Release of Antimicrobial Peptides in Lungs of Virally Infected Mice

Leukotriene B(4) (LTB(4)) is a lipid mediator of inflammation that was recently shown to exert antiviral activities. In this study, we demonstrate that the release of antimicrobial proteins by neutrophils contribute to an early host defense against influenza virus infection in vitro as well as in vivo. Daily i.v. treatments with LTB(4) lead to a significant decrease in lung viral loads at day 5 postinfection in mice infected with influenza A virus compared with the placebo-treated group. This reduction in viral load was not present in mice deficient in the high-affinity LTB(4) receptor. Viral clearance in lungs was associated with up-regulated presence of antimicrobial peptides such as beta-defensin-3, members of the mouse eosinophil-related RNase family, and the mouse cathelicidin-related antimicrobial peptide. Our results also indicate that neutrophils are important in the antiviral effect of LTB(4). Viral loads in neutrophil-depleted mice were not diminished by LTB(4) administration, and a substantial reduction in the presence of murine cathelicidin-related antimicrobial peptide and the murine eosinophil-related RNase family in lung tissue was observed. Moreover, in vitro treatment of human neutrophil cultures with LTB(4) led rapidly to the secretion of the human cathelicidin LL-37 and eosinophil-derived neurotoxin, known as antiviral peptides. Pretreatment of cell cultures with specific LTB(4) receptor antagonists clearly demonstrate the implication of the high-affinity LTB(4) receptor in the LTB(4)-mediated activity. Together, these results demonstrate the importance of neutrophils and the secretion of antimicrobial peptides during the early immune response mediated by LTB(4) against a viral pathogen.

Neutrophil Depletion Causes a Fatal Defect in Murine Pulmonary Staphylococcus aureus clearance

Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare. We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia. Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P < 0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P < 0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight. These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

Global condensation: a "climate change" towards better standardisation of exhaled breath condensate measurements

The clinical response to influenza infection ranges from mild disease to severe pneumonia and it remains unclear whether the inflammatory response to infection is protective or pathogenic. We have defined a novel role for neutrophils in ameliorating lung injury during influenza infection, thereby limiting development of severe disease. Infection of neutrophil-depleted mice with influenza virus HKx31 (H3N2) led to rapid weight loss, pneumonia, and death. Neutropenia was associated with enhanced virus replication in the respiratory tract; however, viral titers were declining at the time of death, leading us to investigate other factors contributing to mortality. In addition to thymic atrophy, lymphopenia, and viremic spread, depletion of neutrophils led to exacerbated pulmonary inflammation, edema, and respiratory dysfunction. Thus, while it is well established that neutrophils contribute to lung injury in a range of pathological conditions, reduced numbers or impaired neutrophil function can facilitate progression of mild influenza to severe clinical disease.

Neutrophils Contribute to Development of a Protective Immune Response during Onset of Infection withLeishmania donovani

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection with Leishmania donovani was investigated in this study. When L. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4(+) and CD8(+) T cells. Increased production of serum IL-4 and IL-10 and higher levels of Leishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response against L. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

The role of ICAM-1 in endotoxin-induced acute renal failure

The pathogenesis of acute renal failure (ARF) occurring during the course of sepsis is incompletely understood. Intercellular adhesion molecule-1 (ICAM-1) is a key cell adhesion molecule upregulated by LPS, which binds to the integrins CD11a/CD18 and CD11b/CD18 present on the surface of leukocytes. We hypothesized that ICAM-1 facilitates renal injury in LPS-induced ARF. To test this, three groups of mice (n = 8 per group) were injected intraperitoneally with 6 mg/kg LPS: 1) normal C57BL/6 mice, 2) mice with a targeted deficiency of ICAM-1 (ICAM-1(-/-)), and 3) mice expressing very low levels of CD18 (CD18-def). ICAM-1(-/-) mice were significantly resistant to LPS-mediated ARF, as opposed to CD18-def mice, which developed severe ARF, as did wild-type controls (48 h blood urea nitrogen 143 +/- 31.5, 70.8 +/- 24.4, and 185 +/- 16.6 mg/dl in wild-type, ICAM-1(-/-), and CD18-def mice, respectively, P < 0.05). At death, ICAM-1(-/-) mice had significantly less renal neutrophil infiltration than the other two groups, as well as less histological tubular injury. Depletion of neutrophils with mAb Gr-1 led to a profound exaggeration of tumor necrosis factor (TNF) release and high mortality, but neutrophil-depleted mice receiving 10-fold less LPS were protected against ARF despite TNF release similar to what is normally associated with LPS-induced ARF. LPS caused a significant increase in renal expression of chemokines; however, this increase was significantly exaggerated in CD18-def mice, which may account for their lack of protection. In conclusion, these data show that ICAM-1 plays a key role in LPS-induced ARF.

A Critical Role for Neutrophils in Resistance to Experimental Infection withBurkholderia pseudomallei

Inhalation is an important route of infection with Burkholderia pseudomallei, the causative agent of melioidosis. In resistant C57BL/6 mice, activated neutrophils are rapidly recruited to the lungs after intranasal B. pseudomallei infection. Prevention of this response by use of the anti-Gr-1+ cell-depleting monoclonal antibody RB6-8C5 severely exacerbated disease, resulting in an acute lethal infection associated with a 1000-fold increase in lung bacterial loads within 4 days. C57BL/6 interferon (IFN)-gamma(-/-) mice were also acutely susceptible to pulmonary B. pseudomallei infection, dying within 3 days of challenge; this suggests that IFN-gamma is essential for control in the lungs and precedes the protective role of neutrophils in resistance. In neutrophil-depleted mice, lung concentrations of tumor necrosis factor (TNF)-alpha, IFN-gamma, and interleukin-6 were decreased by up to 98%. Natural killer cells were the principle source of IFN-gamma, and monocytes were the principle source of TNF-alpha, suggesting that neutrophils play an important indirect role in the generation of the early cytokine environment in the lungs.

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.

Both Influenza-Induced Neutrophil Dysfunction and Neutrophil-Independent Mechanisms Contribute to Increased Susceptibility to a SecondaryStreptococcus pneumoniaeInfection

Since secondary Streptococcus pneumoniae infections greatly increase the mortality of influenza infections, we determined the relative roles of neutrophil-dependent and -independent mechanisms in increased susceptibility to S. pneumoniae during influenza infection. Mice infected with influenza for 6 days, but not 3 days, showed a significant increase in susceptibility to S. pneumoniae infection compared to mice not infected with influenza. There was significant neutrophil accumulation in the lungs of S. pneumoniae-infected mice regardless of whether or not they were infected with influenza for 3 or 6 days. Depletion of neutrophils in these mice resulted in increased susceptibility to S. pneumoniae in both the non-influenza-infected mice and mice infected with influenza for 3 days but not in the mice infected with influenza for 6 days, indicating that a prior influenza infection of 6 days may compromise neutrophil function, resulting in increased susceptibility to a S. pneumoniae infection. Neutrophils from the lungs of mice infected with influenza for 3 or 6 days exhibited functional impairment in the form of decreased phagocytosis and intracellular reactive oxygen species generation in response to S. pneumoniae. In addition, neutrophil-depleted mice infected with influenza for 6 days were more susceptible to S. pneumoniae than neutrophil-depleted mice not infected with influenza, indicating that neutrophil-independent mechanisms also contribute to influenza-induced increased susceptibility to S. pneumoniae. Pulmonary interleukin-10 levels were increased in coinfected mice infected with influenza for 6 days but not 3 days. Thus, an influenza infection of 6 days increases susceptibility to S. pneumoniae by both suppression of neutrophil function and by neutrophil-independent mechanisms such as enhanced cytokine production.

The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection

The urinary tract functions in close proximity to the outside environment, yet must remain free of microbial colonization to avoid disease. The mechanisms for establishing an antimicrobial barrier in this area are not completely understood. Here, we describe the production and function of the cathelicidin antimicrobial peptides LL-37, its precursor hCAP-18 and its ortholog CRAMP in epithelial cells of human and mouse urinary tract, respectively. Bacterial contact with epithelial cells resulted in rapid production and secretion of the respective peptides, and in humans LL-37/hCAP-18 was released into urine. Epithelium-derived cathelicidin substantially contributed to the protection of the urinary tract against infection, as shown using CRAMP-deficient and neutrophil-depleted mice. In addition, clinical E. coli strains that were more resistant to LL-37 caused more severe urinary tract infections than did susceptible strains. Thus, cathelicidin seems to be a key factor in mucosal immunity of the urinary tract.

ANIT toxicity toward mouse hepatocytes in vivo is mediated primarily by neutrophils via CD18

Alpha-naphthylisothiocyanate (ANIT) is a hepatotoxicant that causes acute cholestatic hepatitis with infiltration of neutrophils around bile ducts and necrotic hepatocytes. The objective of this study was to determine whether the beta2-integrin CD18, which plays an important role in leukocyte invasion and cytotoxicity, contributes to ANIT-induced hepatic inflammation and liver injury. Mice with varying levels of leukocyte CD18 expression were treated with ANIT and monitored for hepatic neutrophil influx and liver injury over 48 h. Mice that were partially deficient in CD18 (30% of normal levels) developed periportal inflammation and widespread hepatic necrosis after ANIT treatment in a pattern identical to that in wild-type (WT) mice. In contrast, mice that completely lack CD18 (CD18 null) were resistant to ANIT toxicity. Forty-eight hours after ANIT, CD18-null mice displayed 60% lower serum alanine aminotransferase (ALT) levels and 75% less hepatic necrosis, as shown by morphometry, than WT mice. This was true despite evidence that ANIT still provoked hepatic neutrophil influx in CD18-null mice. WT mice could also be protected from ANIT-induced hepatocellular necrosis, by depleting the animals of neutrophils. Notably, neither CD18-null mice nor neutrophil-depleted WT mice exhibited any attenuation of bile duct injury or cholestasis due to ANIT. We conclude from these experiments that neutrophils invade ANIT-treated livers in a CD18-independent fashion but utilize CD18 to induce hepatocellular cytotoxicity. The results emphasize that neutrophil-mediated amplification of ANIT-induced liver injury is directed toward hepatocytes rather than cholangiocytes. In fact, the data indicate that the majority of ANIT toxicity toward hepatocytes in vivo is neutrophil driven.

Neutrophils regulate the expression of cytokines, chemokines and nitric oxide synthase/nitric oxide in mice injected with Bothrops atrox venom

Bothrops atrox crude venom injected intraperitoneal (i.p.) into BALB/c mice induced local afflux of inflammatory cells, one neutrophil-rich peak after 6 h and another macrophage-rich peak after 48 h. A similar pattern of local cell afflux plus edema, Delta lesions of some skeletal muscle cells, and hemorrhage were observed in mice intramuscular (i.m.) injected with the venom. Measurement of serum cytokines in neutrophil-depleted (by anti-mouse rat monoclonal antibody (mAb) RB6-8C5) and non-depleted BALB/c mice was performed by ELISA. With the exception of IL-1 β (78 pg/ml), higher levels of IL-6 (1348 pg/ml), MIP-1 β (437 pg/ml) and MIP-2 (904 pg/ml) were observed in neutrophil-depleted mice, in comparison to the values found in non-neutrophil depleted mice: IL-1 β (437 pg/ml), IL-6 (750 pg/ml), MIP-1 β (165 pg/ml) and MIP-2 (90 pg/ml). TNF- α was not detected. NO was detected (18 μM) 24 h after venom injection in neutrophil-depleted mice. RT-PCR using representative primers detected expression of mRNA in cells from BALB/c mice injected with B. atrox venom: (a) for IL-1 β, IL-6, inducible nitric oxide synthase (iNOS), CXCR2, MIP-2 and RANTES in cells from mice that were neutrophil-depleted or not; (b) for CCR1, CCR5 and MIP-1 β in cells from neutrophil-depleted mice; (c) for MIP-1 α in cells from non-neutrophil-depleted mice; (d) TNF- α and TGF- β were not detected in either of the mice. These results indicate that neutrophils play a role in regulating the production of some cytokines and chemokines as well as locally expressed or liberated iNOS/NO in tissues injected with B. atrox crude venom.

Accelerated Wound Healing of Alkali-Burned Corneas in MRL Mice Is Associated with a Reduced Inflammatory Signature

The present study was conducted to investigate healing of alkali-burned corneas in MRL/MpJ (MRL) mice. Gross, clinical, and histologic criteria were used to compare healing of alkali-burned corneas in MRL and control C57BL/6J (B6) mice. Effects of neutrophil depletion of B6 mice and allogeneic reconstitution of B6 mice with MRL bone marrow on wound healing were evaluated. Gene expression patterns in normal and wounded corneas were surveyed with array-based quantitative real-time RT-PCR (AQPCR). MRL mice showed accelerated reepithelialization and decreased corneal opacity compared with B6 mice after alkali wounding. Marked inflammatory cell infiltration and fibrosis were evident in the corneas and anterior chambers of B6 mice. MRL mice showed less severe lesions, except for stromal edema. Rapid reepithelialization and reduced keratitis/iritis were also observed in neutrophil-depleted B6 mice, but not in B6 mice reconstituted with MRL bone marrow. AQPCR showed transcriptional changes of fewer genes associated with inflammation and corneal tissue homeostasis in alkali-burned corneas from MRL mice. Increased expression of an anti-inflammatory gene, Socs1, and a gene associated with healing, Mmp1a, were evident in MRL corneas. Alkali-burned corneas heal faster and more completely in MRL mice than in B6 mice, by means of rapid reepithelialization, reduced inflammation, and reduced fibrosis. Reduced inflammation, including decreased neutrophil infiltrates and the lack of a robust proinflammatory gene expression signature correlates with the rapid healing. However, the rapid-healing phenotype is not intrinsic to MRL hematopoietic progenitor cells.

CpG‐oligodeoxynucleotides induce mobilization of hematopoietic progenitor cells into peripheral blood in association with mouse KC (IL‐8) production

The immune system of vertebrates detects bacterial DNA as a "danger signal" based on the presence of unmethylated CpG motifs. We examined whether oligodeoxynucleotides (ODNs) with CpG motifs (CpG-ODNs) also induce mobilization of hematopoietic progenitor cells (HPCs). Mice challenged with CpG-ODNs showed an increase in peripheral blood colony-forming units (CFU) with a peak at day 4 after treatment, associated with an increase, starting 30 min after CpG treatment, in serum levels of mouse keratinocyte-derived chemokine (mKC), a functional homolog of human interleukin (IL) 8; production of granulocyte-colony-stimulating factor (CSF) was also detected. Mobilization and mKC induction were sequence-specific and dose-dependent occurring even with low doses of CpG-ODNs. Interestingly, intestinal cells were involved in mKC production. HPC mobilization by CpG-ODNs was dependent on peripheral blood mononuclear cells since mobilization was reduced in neutrophil-depleted mice. Moreover, CpG-ODN treatment significantly increased G-CSF mobilizing capacity. Finally, pretreatment with an anti-mKC neutralizing antibody significantly reduced CpG-induced mobilization, further supporting a role for mKC. Thus, bacterial DNA is a "danger signal" not only for immune cells but also for hematopoietic cells, communicating the need for increased hematopoiesis during infections and for the renewal of the immune system. The HPC mobilization activity of CpG-ODNs will need to be considered in the design of treatment regimens for cancer clinical trials using CpG-ODNs in association with chemotherapy.

A novel accessory role of neutrophils in concanavalin A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis has been investigated as a model of T cell-mediated liver injury, in which IFN-γ plays an essential role by inducing apoptosis of liver cells. Since a large number of neutrophils infiltrate into the liver in the model, the role of neutrophils was investigated in this study. Con A hardly caused liver injury in neutrophil-depleted mice, as assessed as to the plasma alanine aminotransferase level as well as histochemistry. Neutrophil-depleted mice also failed to produce IFN-γ. Intracellular IFN-γ staining revealed that, among liver leukocytes, T and NK cells but not neutrophils are the main producers of IFN-γ. Nylon wool-purified “T cells,” however, failed to produce IFN-γ in response to Con A in vitro, while the production was restored by the addition of neutrophils. Overall, this study suggests that neutrophils play a novel accessory role in IFN-γ production in Con A-induced hepatitis.

Neutrophil-derived MMP-9 mediates synergistic mobilization of hematopoietic stem and progenitor cells by the combination of G-CSF and the chemokines GROβ/CXCL2 and GROβT /CXCL2Δ4

AbstractMobilized peripheral blood stem cells (PBSCs) are widely used for transplantation, but mechanisms mediating their release from marrow are poorly understood. We previously demonstrated that the chemokines GROβ/CXCL2 and GROβT/CXCL2Δ4 rapidly mobilize PBSC equivalent to granulocyte colony-stimulating factor (G-CSF) and are synergistic with G-CSF. We now show that mobilization by GROβ/GROβT and G-CSF, alone or in combination, requires polymorphonuclear neutrophil (PMN)–derived proteases. Mobilization induced by GROβ/GROβT is associated with elevated levels of plasma and marrow matrix metalloproteinase 9 (MMP-9) and mobilization and MMP-9 are absent in neutrophil-depleted mice. G-CSF mobilization correlates with elevated neutrophil elastase (NE), cathepsin G (CG), and MMP-9 levels within marrow and is partially blocked by either anti–MMP-9 or the NE inhibitor MeOSuc-Ala-Ala-Pro-Val-CMK. Mobilization and protease accumulation are absent in neutrophil-depleted mice. Synergistic PBSC mobilization observed when G-CSF and GROβ/GROβT are combined correlates with a synergistic rise in the level of plasma MMP-9, reduction in marrow NE, CG, and MMP-9 levels, and a coincident increase in peripheral blood PMNs but decrease in marrow PMNs compared to G-CSF. Synergistic mobilization is completely blocked by anti–MMP-9 but not MeOSuc-Ala-Ala-Pro-Val-CMK and absent in MMP-9–deficient or PMN-depleted mice. Our results indicate that PMNs are a common target for G-CSF and GROβ/GROβT-mediated PBSC mobilization and, importantly, that synergistic mobilization by G-CSF plus GROβ/GROβT is mediated by PMN-derived plasma MMP-9.

Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection.

Type I inflammatory cytokines are essential for immunity to many microbial pathogens, including Toxoplasma gondii. Dendritic cells (DC) are key to initiating type 1 immunity, but neutrophils are also a source of chemokines and cytokines involved in Th1 response ignition. We found that T. gondii triggered neutrophil synthesis of CC chemokine ligand (CCL)3, CCL4, CCL5, and CCL20, chemokines that were strongly chemotactic for immature DC. Moreover, supernatants obtained from parasite-stimulated polymorphonuclear leukocytes induced DC IL-12(p40) and TNF-alpha production. Parasite-triggered neutrophils also released factors that induced DC CD40 and CD86 up-regulation, and this response was dependent upon parasite-triggered neutrophil TNF-alpha production. In vivo evidence that polymorphonuclear leukocytes exert an important influence on DC activation was obtained by examining splenic DC cytokine production following infection of neutrophil-depleted mice. These animals displayed severely curtailed splenic DC IL-12 and TNF-alpha production, as revealed by ex vivo flow cytometric analysis and in vitro culture assay. Our results reveal a previously unrecognized regulatory role for neutrophils in DC function during microbial infection, and suggest that cross-talk between these cell populations is an important component of the innate immune response to infection.

Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom.

Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin I, a phospholipase A2 isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin I, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. asper venom and myotoxin I, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative response.