Static and dynamic lengths of neutrophil microvilli

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both ≈0.3 μm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is ≤34 pN (± 3 pN), the length of the microvillus will be extended; if the force is >61 pN (± 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of ≈43 pN/μm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.

Resistance to endotoxic shock and reduced neutrophil migration in mice deficient for the Src-family kinases Hck and Fgr

Signal transduction through the leukocyte integrins is required for the processes of firm adhesion, activation, and chemotaxis of neutrophils during inflammatory reactions. Neutrophils isolated from knockout mice that are deficient in the expression of p59/61hck (Hck) and p58c-fgr (Fgr), members of the Src-family of protein tyrosine kinases, have been shown to be defective in adhesion mediated activation. Cells from these animals have impaired induction of respiratory burst and granule secretion following plating on surfaces that crosslink β2 and β3 integrins. To determine if the defective function of hck−/−fgr−/− neutrophils observed in vitro also results in impaired inflammatory responses in vivo, we examined responses induced by lipopolysaccharide (LPS) injection in these animals. The hck−/−fgr−/− mice showed marked resistance to the lethal effects of high-dose LPS injection despite the fact that high levels of serum tumor necrosis factor α and interleukin 1α were detected. Serum chemistry analysis revealed a marked reduction in liver and renal damage in mutant mice treated with LPS, whereas blood counts showed a marked neutrophilia that was not seen in wild-type animals. Direct examination of liver sections from mutant mice revealed reduced neutrophil migration into the tissue. These data demonstrate that defective integrin signaling in neutrophils, caused by loss of Hck and Fgr tyrosine kinase activity, results in impaired inflammation-dependent tissue injury in vivo.

An opsonic function of the neutrophil bactericidal/permeability-increasing protein depends on both its N- and C-terminal domains

The host response to Gram-negative bacterial infection is influenced by two homologous lipopolysaccharide (LPS)-interactive proteins, LPS-binding protein (LBP) and the bacteridical/permeability-increasing protein (BPI). Both proteins bind LPS via their N-terminal domains but produce profoundly different effects: BPI and a bioactive N-terminal fragment BPI-21 exert a selective and potent antibacterial effect upon Gram-negative bacteria and suppress LPS bioactivity whereas LBP is not toxic toward Gram-negative bacteria and potentiates LPS bioactivity. The latter effect of LBP requires the C-terminal domain for delivery of LPS to CD14, so we postulated that the C-terminal region of BPI may serve a similar delivery function but to distinct targets. LBP, holoBPI, BPI-21, and LBP/BPI chimeras were compared for their ability to promote uptake by human phagocytes of an encapsulated, phagocytosis-resistant strain of Escherichia coli. We show that only bacteria preincubated with holoBPI are ingested by neutrophils and monocytes. These findings suggest that, when extracellular holoBPI is bound via its N-terminal domain to Gram-negative bacteria, the C-terminal domain promotes bacterial attachment to neutrophils and monocytes, leading to phagocytosis. Therefore, analogous to the role of the C-terminal domain of LBP in delivery of LPS to CD14, the C-terminal domain of BPI may fulfill a similar function in BPI-specific disposal pathways for Gram-negative bacteria.

Cytokine-mediated Bax deficiency and consequent delayed neutrophil apoptosis: A general mechanism to accumulate effector cells in inflammation

Neutrophils are important effector cells in immunity to microorganisms, particularly bacteria. Here, we show that the process of neutrophil apoptosis is delayed in several inflammatory diseases, suggesting that this phenomenon may represent a general feature contributing to the development of neutrophilia, and, therefore, in many cases to host defense against infection. The delay of neutrophil apoptosis was associated with markedly reduced levels of Bax, a pro-apoptotic member of the Bcl-2 family. Such Bax-deficient cells were also observed upon stimulation of normal neutrophils with cytokines present at sites of neutrophilic inflammation, such as granulocyte and granulocyte–macrophage colony-stimulating factors, in vitro. Moreover, Bax-deficient neutrophils generated by using Bax antisense oligodeoxynucleotides demonstrated delayed apoptosis, providing direct evidence for a role of Bax as a pro-apoptotic molecule in these cells. Interestingly, the Bax gene was reexpressed in Bax-deficient neutrophils under conditions of cytokine withdrawal. Thus, both granulocyte expansion and the resolution of inflammation appear to be regulated by the expression of the Bax gene in neutrophils.

Modes of action of aspirin-like drugs: Salicylates inhibit Erk activation and integrin-dependent neutrophil adhesion

The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness of formylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1–8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion by formylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses.

Production of β-defensins by human airway epithelia

Human β-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1β stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.

Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67PHOX and cytochrome b558

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac1 or Rac2, with the membrane-bound cytochrome b558. Whereas the interaction of p47PHOX with cytochrome b558 is well established, an interaction between p67PHOX and cytochrome b558 has never been investigated. We report here a direct interaction between p67PHOX and cytochrome b558. First, labeled p67PHOX recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b558-deficient patient. Second, p67PHOX binds to cytochrome b558 that has been bound to nitrocellulose. Third, GTP-p67PHOX bound to glutathione agarose is able to pull down cytochrome b558. Rac1-GTP or Rac1-GDP increased the binding of p67PHOX to cytochrome b558, suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67PHOX and cytochrome b558.

Dissipative metabolic patterns respond during neutrophil transmembrane signaling

Self-organization is a common theme in biology. One mechanism of self-organization is the creation of chemical patterns by the diffusion of chemical reactants and their nonlinear interactions. We have recently observed sustained unidirectional traveling chemical redox [NAD(P)H − NAD(P)+] waves within living polarized neutrophils. The present study shows that an intracellular metabolic wave responds to formyl peptide receptor agonists, but not antagonists, by splitting into two waves traveling in opposite directions along a cell's long axis. Similar effects were noted with other neutrophil-activating substances. Moreover, when cells were exposed to an N-formyl-methionyl-leucyl-phenylalanine (FMLP) gradient whose source was perpendicular to the cell's long axis, cell metabolism was locally perturbed with reorientation of the pattern in a direction perpendicular to the initial cellular axis. Thus, extracellular activating signals and the signals' spatial cues are translated into distinct intracellular dissipative structures.

Different functions for the interleukin 8 receptors (IL-8R) of human neutrophil leukocytes: NADPH oxidase and phospholipase D are activated through IL-8R1 but not IL-8R2.

Two monoclonal antibodies, anti-IL8R1 and anti-IL8R2, raised against both interleukin 8 receptors (IL-8R) of human neutrophils, IL-8R1 and IL-8R2, were used to study individual receptor functions after stimulation with IL-8, GRO alpha, or NAP-2. Efficacy and selectivity of the antibodies were tested in Jurkat cells transfected with cDNA coding for one or the other receptor. The binding of 125 I labeled IL-8 and IL-8-induced changes of the cytosolic free Ca2+ concentration were inhibited by anti-IL8RI in cells expressing IL-8R1 and by anti-IL8R2 in cells expressing IL-8R2. In human neutrophils, release of elastase was observed after stimulation with IL-8 or GRO alpha. The response to IL-8 was inhibited slightly by anti-IL8R1 and more substantially when both monoclonal antibodies were present, while the response to GRO alpha was inhibited by anti-IL8R2 but was not affected by anti-IL8R1. These results indicate that both IL-8 receptors can signal independently for granule enzyme release. Superoxide production, a measure of the respiratory burst, was obtained with increasing concentrations of IL-8 with maximum effects at 25 to 50 nM, but no response was observed upon challenge with GRO alpha or NAP-2 up to 1000 nM. The superoxide production induced by IL-8 was inhibited by anti-IL8R1, but was not affected by anti-IL8R2. Stimulation of neutrophils with IL-8, in contrast to GRO alpha or NAP-2, also elicited phospholipase D activity. The effect of IL-8 was again inhibited by anti-IL-8R1 but not by anti-IL8R2, indicating that this response, like the respiratory burst, was mediated by IL-8R1. Taken together, our results show that IL-8R1 and IL-8R2 are functionally different. Responses, such as cytosolic free Ca2+ changes and the release of granule enzymes, are mediated through both receptors, whereas the respiratory burst and the activation of phospholipase D depend exclusively on stimulation through IL-8R1.

Cell-free activation of neutrophil NADPH oxidase by a phosphatidic acid-regulated protein kinase.

The phosphorylation-dependent mechanisms regulating activation of the human neutrophil respiratory-burst enzyme, NADPH oxidase, have not been elucidated. We have shown that phosphatidic acid (PA) and diacylglycerol (DG), products of phospholipase activation, synergize to activate NADPH oxidase in a cell-free system. We now report that activation by PA plus DG involves protein kinase activity, unlike other cell-free system activators. NADPH oxidase activation by PA plus DG is reduced approximately 70% by several protein kinase inhibitors [1-(5-isoquinolinesulfonyl)piperazine, staurosporine, GF-109203X]. Similarly, depletion of ATP by dialysis reduces PA plus DG-mediated NADPH oxidase activation by approximately 70%. Addition of ATP, but not a nonhydrolyzable ATP analog, to the dialyzed system restores activation levels to normal. In contrast, these treatments have little effect on NADPH oxidase activation by arachidonic acid or SDS plus DG. PA plus DG induces the phosphorylation of a number of endogenous proteins. Phosphorylation is largely mediated by PA, not DG. A predominant substrate is p47-phox, a phosphoprotein component of NADPH oxidase. Phosphorylation of p47-phox precedes activation of NADPH oxidase and is markedly reduced by the protein kinase inhibitors. In contrast, arachidonic acid alone or SDS plus DG is a poor activator of protein phosphorylation in the cell-free system. Thus, PA induces activation of one or more protein kinases that regulate NADPH oxidase activation in a cell-free system. This cell-free system will be useful for identifying a functionally important PA-activated protein kinase(s) and for dissecting the phosphorylation-dependent mechanisms responsible for NADPH oxidase activation.

Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R−/− mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R−/− animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.

Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression

Leukocyte interactions with vascular endothelium during inflammation occur through discrete steps involving selectin-mediated leukocyte rolling and subsequent firm adhesion mediated by members of the integrin and Ig families of adhesion molecules. To identify functional synergy between selectin and Ig family members, mice deficient in both L-selectin and intercellular adhesion molecule 1 (ICAM-1) were generated. Leukocyte rolling velocities in cremaster muscle venules were increased significantly in ICAM-1-deficient mice during both trauma- and tumor necrosis factor α-induced inflammation, but rolling leukocyte flux was not reduced. Elimination of ICAM-1 expression in L-selectin-deficient mice resulted in a sharp reduction in the flux of rolling leukocytes during tumor necrosis factor α-induced inflammation. The observed differences in leukocyte rolling behavior demonstrated that ICAM-1 expression was required for optimal P- and L-selectin-mediated rolling. Increased leukocyte rolling velocities presumably translated into decreased tissue emigration because circulating neutrophil, monocyte, and lymphocyte numbers were increased markedly in L-selectin/ICAM-1-deficient mice. Furthermore, neutrophil emigration during acute peritonitis was reduced by 80% in the double-deficient mice compared with either L-selectin or ICAM-1-deficient mice. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling in vivo, which is essential for the generation of effective inflammatory responses.

Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl− secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl− secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl− secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl− secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl− secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.

Epithelial antibiotic induced in states of disease

Epithelial defensins provide an active defense against the external microbial environment. We investigated the distribution and expression of this class of antimicrobial peptides in normal cattle and in animals in varying states of disease. β-defensin mRNA was found to be widely expressed in numerous exposed epithelia but was found at higher levels in tissues that are constantly exposed to and colonized by microorganisms. We observed induction in ileal mucosa during chronic infection with Mycobacterium paratuberculosis and in bronchial epithelium after acute infection with Pasteurella haemolytica. It has been proposed that expression of antimicrobial peptides is an integral component of the inflammatory response. The results reported here support this hypothesis and suggest that epithelial defensins provide a rapidly mobilized local defense against infectious organisms.

Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans

We have cloned and sequenced two defensins, Smd1 and Smd2, from anterior midgut tissue of the blood-sucking fly Stomoxys calcitrans. The DNA and N-terminal protein sequences suggest both are produced as prepropeptides. Smd1 differs from the classic defensin pattern in having an unusual six-amino acid-long N-terminal sequence. Both Smd1 and Smd2 have lower pI points and charge than insect defensins derived from fat body/hemocytes. Northern analysis shows both of these defensin molecules are tissue specific; both are produced by the anterior midgut tissue and, unlike the other insect defensins reported to date, neither appears to be expressed in fat body or hemocytes. Northern analysis also shows that mRNAs for both defensins are constitutively produced in the anterior midgut tissues and that these transcripts are up-regulated in response to sterile as well as a lipopolysaccharide-containing blood meal. However, anti-Gram-negative biological activity in the midgut is substantially enhanced by lipopolysaccharide. These findings suggest that the insect midgut has its own tissue-specific immune mechanisms and that this invertebrate epithelium is, like several vertebrate epithelia, protected by specific antibacterial peptides.