PMN Emigration Research Articles

Interleukin-23-Mediated Inflammation in Pseudomonas aeruginosa Pulmonary Infection

Pseudomonas aeruginosa is an opportunistic pathogen that is capable of causing acute and chronic pulmonary infection in the immunocompromised host. In the case of cystic fibrosis (CF), chronic P. aeruginosa infection causes increased mortality by promoting overly exuberant airway inflammation and cumulative lung damage. Identifying the key regulators of this inflammation may lead to the development of new therapies that improve P. aeruginosa-related mortality. We report here that interleukin-23 (IL-23), the cytokine most clearly tied to IL-17-mediated inflammation, also promotes IL-17-independent inflammation during P. aeruginosa pulmonary infection. During the early innate immune response, prior to IL-17 induction, IL-23 acts synergistically with IL-1β to promote early neutrophil (polymorphonuclear leukocyte [PMN]) recruitment. However, at later time points, IL-23 also promoted IL-17 production by lung γδ T cells, which was greatly augmented in the presence of IL-1β. These studies show that IL-23 controls two independent phases of neutrophil recruitment in response to P. aeruginosa infection: early PMN emigration that is IL-17 independent and later PMN emigration regulated by IL-17.

A Chemotactic Peptide from Laminin α5 Functions as a Regulator of Inflammatory Immune Responses via TNFα-mediated Signaling

Tissue injury triggers inflammatory responses that may result in release of degradation products or exposure of cryptic domains of extracellular matrix components. Previously, we have shown that a cryptic peptide (AQARSAASKVKVSMKF) in the alpha-chain of laminin-10 (alpha5beta1gamma1), a prominent basement membrane component, is chemotactic for both neutrophils (PMNs) and macrophages (Mphis) and induces matrix metalloproteinase-9 (MMP-9) production. To determine whether AQARSAASKVKVSMKF has additional effects on inflammatory cells, we performed microarray analysis of RNA from RAW264.7 Mphis stimulated with AQARSAASKVKVSMKF. Several cytokines and cytokine receptors were increased >3-fold in response to the laminin alpha5 peptide. Among these were TNF-alpha and one of its receptors, the p75 TNFR (TNFR-II), increasing 3.5- and 5.7-fold, respectively. However, the peptide had no effect on p55 TNFR (TNFR-I) expression. Corroborating the microarray data, the protein levels of TNF-alpha and TNFR-II were increased following stimulation of RAW264.7 cells with AQARSAASKVKVSMKF. In addition, we determined that the production of TNF-alpha and TNFR-II in response to AQARSAASKVKVSMKF preceded the production of MMP-9. Furthermore, using primary Mphis from mice deficient in TNFR-I, TNFR-II, or both TNF-alpha receptors (TNFRs), we determined that AQARSAASKVKVSMKF induces MMP-9 expression by Mphis through a pathway triggered by TNFR-II. However, TNF-alpha signaling is not required for AQARSAASKVKVSMKF-induced PMN release of MMP-9 or PMN emigration. These data suggest that interactions of inflammatory cells with basement membrane components may orchestrate immune responses by inducing expression of cytokines, recruitment of inflammatory cells, and release of proteinases.

Tumor necrosis factor receptor 2 contributes to ozone-induced airway hyperresponsiveness in mice.

The purpose of this study was to determine whether tumor necrosis factor (TNF) contributes to airway hyperresponsiveness (AHR) and migration of polymorphonuclear leukocytes (PMN) into the airways following exposure to ozone (O(3)). Wild-type mice, TNF p55 or p75 receptor knockout mice (p55 TNFR -/- and p75 TNFR -/-), as well as double receptor knockout mice (p55/p75 TNFR -/-), were exposed to O(3). Three hours after cessation of O(3), airway responses to inhaled methacholine were determined by whole body plethysmography using changes in enhanced pause (Penh) as an index of airway narrowing. In wild-type mice, O(3) exposure (0.5 ppm, 3 h) caused a significant increase in airway responsiveness as indicated by a 1.2 log leftward shift in the methacholine dose- response curve. In contrast, in p55/p75 TNFR -/- mice, O(3) caused only a 0.5 log shift in the dose-response curve (p < 0.05 compared with wild-type). Similar results were obtained in p75 TNFR -/- mice. In contrast, O(3)-induced airway hyperresponsiveness was not different in WT and p55 TNFR -/- mice. During O(3) exposure (1 pm, 3 h), minute ventilation (V E) decreased by 64 +/- 4% in wild-type, but only 24 +/- 5% in p55/p75 TNFR -/- mice, indicating that despite their reduced O(3)-induced AHR, the TNFR-deficient mice actually inhaled a greater dose of O(3). Similar results were obtained in p75 -/- mice, whereas changes in V E induced by O(3) were the same in wild-type and p55 -/- mice. PMN numbers in bronchoalveolar lavage fluid recovered 21 h after cessation of exposure to O(3) (2 ppm, 3 h) were significantly increased compared with after air exposure but were not different in wild-type and p55/p75 TNFR -/- mice. Our results indicate that TNF contributes to the AHR but not the PMN emigration induced by acute O(3) exposure. whole body plethysmography; polymorphonuclear leukocytes; minute ventilation; knockout mice; methacholine

Role of 5-Lipoxygenase Products in the Local Accumulation of Neutrophils in Dermal Inflammation in the Rabbit

AbstractStudies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5–300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-α, IL-1β, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.

Gelatinase B Deficiency Does Not Protect Against Lipopolysaccharide-Induced Acute Lung Injury

Polymorphonuclear leukocytes (PMNs) contain gelatinase B, a matrix metalloproteinase that is readily released from these cells upon stimulation with a variety of agents. Because gelatinase B degrades basement membrane components, and because inhibition of matrix metalloproteinase activity blunts PMN migration through basement membrane in vitro, gelatinase B may be required for PMN emigration in the lung and other tissues. High levels of gelatinase B have been reported in BAL fluid in ARDS patients, raising the possibility that gelatinase B contributes to the pathophysiology of ARDS.

Neutrophil emigration in the lungs, peritoneum, and skin does not require gelatinase B.

Polymorphonuclear leukocytes (PMN) release gelatinase B in response to variable stimuli. Gelatinase B degrades basement membrane components in vitro, and inhibition of matrix metalloproteinase activity blunts PMN migration through a prototype basement membrane (Matrigel) and amnionic membranes. Accordingly, it has been speculated that gelatinase B is necessary for PMN emigration. To test this hypothesis we induced acute inflammation in the lungs, peritoneum, and skin in mice with a null mutation of the gelatinase B gene (gelatinase B-/-) and littermate controls (gelatinase B+/+). At 3, 6, 12, and 24 h after intratracheal instillation of LPS, the emigration of PMN in the lung, as determined by PMN in bronchoalveolar lavage fluid, was similar in gelatinase B-/- and gelatinase B+/+ mice. The number of PMN in the peritoneal cavity 4 h after thioglycollate-induced peritonitis was also comparable in gelatinase B-/- and gelatinase B+/+ mice. At 4 h after an intradermal injection of interleukin-8, numerous PMN were present extravascularly in the dermis in both gelatinase B-/- and gelatinase B+/+ mice and the myeloperoxidase activities of the skin at the injection sites were indistinguishable between the two types of mice. PMN from gelatinase B-/- mice migrated through Matrigel in response to zymosan-activated serum with the same efficiency as did PMN from gelatinase B+/+ mice. In vitro, gelatinase B-/- PMN killed Staphylococcus aureus and Klebsiella pneumoniae as effectively as did PMN from gelatinase B+/+ mice. These findings indicate that gelatinase B is not required for PMN emigration, and suggest that the antibacterial function of PMN is preserved despite gelatinase B deficiency.

The Role of Leukocyte Emigration and IL-8 on the Development of Lipopolysaccharide-Induced Lung Injury in Rabbits

Leukocyte emigration and alveolar macrophage-derived cytokines may contribute to lung microvascular injury associated with adult respiratory distress syndrome. We have used mAbs against cell adhesion molecules on leukocytes (anti-CD18 and anti-CD49d) or against IL-8 to investigate these contributions. Intratracheal (i.t.) instillation of LPS (50 microg/kg) caused a significant increase in bronchoalveolar lavage polymorphonuclear leukocytes (PMNs) without an increase in mononuclear cells (MNCs) or an increase in lung permeability. Injection of LPS (10 microg/kg) i.v. at 24 h after i.t. LPS caused significant increases in bronchoalveolar lavage PMNs, MNCs, IL-8, and monocyte chemotactic protein-1, as well as increases in lung permeability. Rabbits that were administered i.t. LPS followed by i.v. LPS and treated with anti-CD18 mAb had a significantly lower lung permeability index and emigration of fewer PMNs but no change in MNC emigration compared with saline treatment. Anti-IL-8 mAb treatment resulted in a significantly lower lung permeability index with no change in PMN emigration compared with no treatment. These results suggest that PMN emigration is necessary but not sufficient for the development of LPS-induced lung injury, and that IL-8 plays a significant role in PMN-dependent lung injury, independent of PMN emigration.

Role of lipopolysaccharide in signaling to subepithelial polymorphonuclear leukocytes.

Polymorphonuclear leukocyte (PMN) infiltration and migration across colonic intestinal epithelia is a hallmark of inflammation in Shigella flexneri-mediated dysentery. To identify bacterial signals associated with this process, potential stimulatory factors mediating initial PMN association with the epithelium and subsequent transepithelial migration were examined in an in vitro model system. Quantitative analyses revealed that purified S. flexneri lipopolysaccharide (LPS) deposited at the apical surface of polarized intestinal epithelial cells transcytosed to the basolateral pole, a process dependent on the stage of epithelial cell differentiation. Transcytosed LPS in the presence of normal human serum (NHS), a source of LPS binding protein and soluble CD14, mediated both interleukin-8 secretion at the basolateral pole and enhanced PMN adherence. In addition, LPS stimulated a significant degree of directed transepithelial migration of PMNs, an event that was further enhanced in the presence of NHS. These results implicate LPS in signaling subepithelial PMN emigration and enhancing PMN-epithelium interactions prior to and during subsequent Shigella-induced transepithelial migration.

Endothelial activation in monosodium urate monohydrate crystal-induced inflammation: in vitro and in vivo studies on the roles of tumor necrosis factor alpha and interleukin-1.

There is relatively little direct evidence for the roles of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in activating endothelium in vivo. The aim of this study was to use in vitro and in vivo models to investigate the contribution of these cytokines to both E-selectin expression and the recruitment of polymorphonuclear cells (PMN) in monosodium urate monohydrate (MSU) crystal-induced inflammation. MSU crystals were incubated with freshly isolated mononuclear cells, after which the harvested supernatants were tested for their ability to induce E-selectin expression during coculture with human umbilical vein endothelial cells. Subsequent experiments were performed with the addition of neutralizing anticytokine antibodies/antisera. The role of TNF alpha was then studied in an MSU crystal-induced monarthritis model, in the presence or absence of anti-TNF alpha (5 mg/kg intravenously). 99mtechnetium (99mTc)-labeled PMN cells and (111)indium (111In)-labeled anti-E-selectin monoclonal antibody (MAb) 1.2B6 were intravenously administered 4 hours after intraarticular injection to quantify PMN recruitment and E-selectin expression in inflamed joints. MSU crystals were a potent stimulus for IL-1 and TNF alpha production by monocytes in vitro, and these cytokines fully accounted for MSU crystal-stimulated, monocyte-mediated endothelial activation. In the MSU crystal-induced monarthritis model, TNF alpha blockade was very effective in suppressing both E-selectin expression and PMN emigration into the inflamed joints, as judged by gamma-camera image analysis and postmortem tissue counting following the intravenous injection of 99mTc-PMN and 111In-anti-E-selectin MAb. IL-1 and TNF alpha appear to be the only factors released by monocytes following incubation with MSU crystals, which induce E-selectin expression in vitro. Anti-TNF alpha is effective in suppressing endothelial activation and PMN recruitment in vivo E-selectin imaging can be used to assess the endothelial response to therapy and may prove useful for clinical studies.

L-selectin (CD62L) blockade does not impair peritoneal neutrophil emigration or subcutaneous host defense to bacteria in rabbits.

Neutrophil (PMN) recruitment into systemic inflammatory sites in vivo is thought to be initiated by selectin-mediated endothelial adherence. We explored the role of L-selectin (CD62L) in leukocyte emigration following instillation of bacteria into the peritoneum or s.c. skin in rabbits. Pretreatment with blocking mAb against L-selectin (LAM1.3) reduced peritoneal PMN emigration 4 h after i.p. inoculation with 10(10) CFU of Escherichia coli by only 17% compared with animals receiving a nonblocking L-selectin mAb (LAM1.14). Peritoneal PMNs from saline-treated rabbits demonstrated a complete absence of L-selectin, whereas those from LAM1.3-treated animals retained 43% of their baseline L-selectin expression. This suggests that L-selectin shedding is not a requisite event for PMN emigration under these conditions. In rabbits given s.c. inoculations with either Staphylococcus aureus or E coli, pretreatment with mAb LAM1.3 did not significantly impair PMN emigration at 24 h, nor increase the incidence, size, or associated mortality of resulting abscesses at 7 days compared with animals receiving nonblocking mAb LAM1.14. We conclude that: 1) mAb blockade of L-selectin in vivo only modestly affects acute, E. coli-induced peritoneal PMN emigration; and 2) L-selectin blockade does not increase infectious sequelae associated with s.c. bacterial inoculation. These findings of only mildly reduced PMN emigration into the peritoneum and no alteration in s.c. host defense differ from those reported with L-selectin blockade under other, nonbacterial inflammatory conditions, and suggest that redundant selectin-mediated mechanisms (P- and E-selectin) are sufficient for normal PMN emigration in response to bacterial stimulation.

Intratracheal injection of endotoxin and cytokines. IX. Contribution of CD11a/ICAM-1 to neutrophil emigration.

Intratracheal injection of endotoxin [lipopolysaccharide (LPS)] in rats causes acute inflammation characterized by the emigration of neutrophils (PMNs) into the bronchoalveolar airspace. Antibody to PMN adhesion molecule CD11a inhibited LPS-initiated PMN accumulation in bronchoalveolar lavage (BAL) fluid by 32% (P < 0.001). Antibody to the endothelial CD11a counterreceptor intercellular adhesion molecule-1 (ICAM-1) inhibited LPS-initiated PMN accumulation in BAL fluid by 66% (P < 0.0001). Combined antibody blockade of ICAM-1 and the C-X-C chemokine cytokine-induced neutrophil chemoattractant (CINC) inhibited LPS-initiated PMN emigration by 80%, significantly more than antibody against either ICAM-1 or CINC alone. To study the relative contribution of alveolar macrophages and PMNs to intra-alveolar tumor necrosis factor (TNF), the LPS-induced TNF in BAL fluid was measured after depletion of circulating PMNs with a cytolytic antibody to CD18. Although the anti-CD18 antibody completely abrogated LPS-initiated PMN emigration into BAL fluid, TNF levels in BAL fluid were unaffected, suggesting that alveolar macrophages are the predominant cellular source of LPS-induced TNF production. In conclusion, 1) CD11a, ICAM-1, and CINC play major roles in the LPS-initiated emigration of PMNs into the bronchoalveolar space, and 2) the TNF that drives ICAM-1 and CINC expression is derived largely from alveolar macrophages rather than PMNs.

Ischemia/reperfusion-induced granulocyte influx is a multistep process mediated by mast cells.

The influx of polymorphonuclear leukocytes (PMNs) that occurs at the onset of reperfusion of postischemic tissue is a multistep mechanism that includes initial rolling of PMNs, a progressive reduction in PMN rolling velocity, and ultimately PMN adhesion and emigration. In this study we assessed whether mast cells play a role in reperfusion-induced PMN recruitment with emphasis on PMN rolling, adhesion, and emigration. Cat small intestinal venules were visualized using intravital microscopy during control, 60 min of ischemia (intestinal blood flow 20% of control), and 60 min of reperfusion in untreated and cromolyn- (mast cell stabilizer) pretreated animals. Between 1 and 5 min of reperfusion, there was a very dramatic rise in PMN rolling which persisted for the next 60 min. In cromolyn-pretreated animals, the flux of rolling PMNs increased in the very early (5 min) reperfusion period, but by 10 min the number of rolling leukocytes was significantly reduced, an event that persisted for the next 60 min. PMNs rolled at a velocity of 40% of control values for the first 10 min of reperfusion without a change in hemodynamic values but returned toward control values by 60 min of reperfusion. An identical velocity profile was observed for the cromolyn-treated animals. In untreated animals, PMN adhesion and emigration was significantly increased throughout the reperfusion period, whereas the cromolyn-pretreated animals had reduced adhesion (60 min of reperfusion) and a reduced rate of emigration throughout the reperfusion period. Interstitial mast cells contribute significantly to the multistep recruitment (rolling, adhesion, and emigration) of PMNs from blood to postischemic tissues and raise the possibility that antiallergy agents such as cromolyn may be a useful form of therapy in the postischemic intestine.

The vascular response in chronic periodontitis.

The role of vascular endothelial cells (EC) in periodontitis was investigated in a series of histological studies. Expansion of the vasculature was found to occur with development of gingivitis and periodontitis. This was thought to contribute to the characteristic tissue degradation in the developing disease. Vascular expansion could also play a role in the formation of a previously unreported perivascular hyaline material (PHyM). Polymorphonuclear leukocytes (PMN) are known to be protective in periodontitis, and the location, incidence and extent of PHyM suggested a role for PHyM in periodontitis by inhibiting PMN emigration. PMN emigration was found to occur from specialized high EC (HEC) lined post capillary venules. This was unexpected, as such vessels have previously been found to exchange lymphocytes almost exclusively. Detailed histochemical, ultrastructural and biosynthetic studies of these specialized blood vessels led to the suggestion that HEC may be specially adapted for the synthesis of cytokines in periodontitis. A negative association between expression of the membrane bound ectoenzyme, alkaline phosphatase, and HEC suggested a role for this enzyme in leukocyte emigration. These observations compel re-evaluation of the role of EC in chronic inflammation, and in periodontitis in particular. The direction of current and future work is discussed.

P-selectin blockade does not impair leukocyte host defense against bacterial peritonitis and soft tissue infection in rabbits.

Adhesion molecules are responsible for PMN-endothelial cell interactions involved in both PMN-mediated endothelial injury (e.g., after ischemia-reperfusion injury) and PMN-mediated host defense against bacterial infection. Inhibition of PMN-endothelial adherence with CD18 and P-selectin mAb has been shown to ameliorate the tissue injury resulting from ischemia and reperfusion under a variety of experimental conditions. However, interference with PMN function may result in an increased risk of bacterial infection. Previous investigations suggest that CD18 blockade can lead to increased infectious risk. Little is known of the infectious risks associated with selectin blockade. We report the effects of P-selectin blockade (using mAb PB1.3) on bacteria-induced PMN emigration into the peritoneum and subcutaneous (s.c.) tissue in rabbits. Leukocyte and PMN emigration into the peritoneum 4 h after inoculation with 10 ml of 10(9) CFU/ml Escherichia coli was significant in saline-treated animals, and not different in animals pretreated with mAb PB1.3. Similarly, the incidence and severity of abscess formation 7 days after s.c. inoculation with Staphylococcus aureus (10(7), 10(8), or 10(9) CFU) was not increased in rabbits pretreated with mAb PB1.3 compared to saline. PMN emigration to the s.c. S. aureus was also similar in both saline and mAb PB1.3-treated animals, as determined by light microscopy. We conclude that P-selectin blockade with mAb PB1.3: 1) does not interfere with acute, E. coli-induced PMN emigration into the peritoneum, 2) does not increase the incidence or severity of S. aureus abscess formation in s.c. tissue, and 3) interferes less with PMN antibacterial host defense mechanisms than inhibition of CD18-mediated PMN adherence.

CD18-independent neutrophil and mononuclear leukocyte emigration into the peritoneum of rabbits.

The CD18 mAb 60.3 and the CD49d mAb HP1/2 were given at the time of intraperitoneal instillation of either protease peptone or live Escherichia coli bacteria and at 12 h. Leukocyte emigration was evaluated at 4 and 24 h. PMN emigration 4 h after protease peptone instillation and injection of both mAbs was 10% of that in saline treatment. It was 15% of that in saline treatment after mAb 60.3 alone and unchanged by mAb HP1/2. At 24 h PMN emigration in response to protease peptone was not prevented by either CD18 or CD49d mAbs, however, when given together emigration was 10% of saline-treated animals. Mononuclear cell emigration to protease peptone was enhanced at 4 h by both CD18 and CD49d mAbs. The CD18 mAb did not augment mononuclear emigration in response to live bacteria. At 24 h, neither the CD18 nor the CD49d mAb alone blocked emigration of mononuclear cells, but the combination of the two did. These studies demonstrate that: (a) early (4 h) PMN emigration is CD11/CD18 dependent; (b) late (24 h) PMN emigration is CD11/CD18 independent; and (c) mononuclear cells utilize the integrins CD18 and CD49d.

Role of protein synthesis and CD11/CD18 adhesion complex in neutrophil emigration into the lung.

The mechanism of neutrophil (PMN) emigration into the lung may be stimulus-dependent. This study examined PMN emigration in the lung induced by intratracheal instillation of lipopolysaccharide (LPS), Streptococcus pneumoniae (S. pneu) organisms, supernatant from S. pneu incubated with alveolar macrophages (AM phi), Escherichia coli (E. coli) organisms, or phorbol myristate acetate (PMA). Rabbits were pretreated with either the CD18 monoclonal antibody (MAb) 60.3, the protein synthesis inhibitor cycloheximide (Cx), or, in one case, both. Animals were then given one of the above stimuli to elicit PMN emigration. Four hours after the stimulus was instilled, animals were killed and total and differential cell counts were performed on bronchoalveolar lavage (BAL) fluid. PMN emigration in response to PMA was virtually abolished by MAb 60.3, but was not significantly inhibited by Cx. Emigration induced by LPS was inhibited by 80% by either MAb 60.3 or Cx, and greater than 94% when MAb 60.3 and Cx were given simultaneously. Emigration in response to E. coli organisms was 80% inhibited by MAb 60.3. Emigration induced by S. pneu was approximately 50% inhibited by MAb 60.3, but was greater than 90% blocked by Cx. The MAb 60.3 had approximately the same effect on PMN emigration toward the supernatant from co-incubation of AM phi with S. pneu as it did toward live S. pneu. It is concluded that the mechanism of PMN emigration into the lung is stimulus-dependent. The CD18-dependent mechanism is responsible for the majority of the emigration in response to PMA, E. coli LPS, and E. coli organisms. S. pneu and supernatant from S. pneu + AM phi produce a CD18-independent pathway. These data suggest the requirement for de novo protein synthesis for PMN emigration in response to LPS and S. pneu, but not for PMA-induced emigration.

Intravascular IL-8. Inhibitor of polymorphonuclear leukocyte accumulation at sites of acute inflammation.

IL-8 has been characterized primarily as a polymorphonuclear leukocyte (PMN) chemoattractant and proinflammatory mediator. Recently, we have reported that [Ala-IL-8]77 is secreted by activated cultured human endothelial cells and can function as a potent inhibitor of PMN adhesion to these monolayers. The pathophysiologic relevance of this in vitro observation was examined by determining the effects of intravascular or extravascular administration of IL-8 on PMN emigration at sites of acute inflammation in the skin of NZW rabbits. An i.v. bolus of [Ala-IL-8]77 (12 micrograms/kg) produced a marked and selective reduction of circulating PMN within 3 min, which returned toward preinjection levels within 30 min, and subsequently exceeded this level. A similar response was observed for circulating radiolabeled PMN, and gamma-scintigraphy determined that the lungs were the primary site of leukosequestration. During the 30- to 150-min interval after i.v. infusion of [Ala-IL-8]77, PMN emigration into acute inflammatory sites, elicited by various chemoattractants or cytokines, was significantly reduced, as judged histologically and quantitated with 51Cr-labeled PMN and myeloperoxidase measurements. Intravenous administration of [Ser-IL-8]72 yielded similar results. This inhibitory effect of i.v. IL-8 was transient and reinducible and did not reflect a suppression of the responsiveness of circulating PMN to chemoattractants. Intradermal injections of [Ala-IL-8]77 or [Ser-IL-8]72 induced dose-dependent PMN accumulation, which also was significantly reduced by i.v. administration of either form of IL-8. These results indicate that i.v. IL-8 can function as a PMN-directed leukocyte adhesion inhibitor and suggest that local secretion of IL-8 by activated endothelium may differentially modulate leukocyte-endothelial interactions at sites of acute inflammation.

CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits.

Neutrophil (PMN) migration in the systemic and pulmonary circulation of rabbits was compared by using different inflammatory stimuli to determine the role of the leukocyte adhesion complex, CD11/CD18, in each of these vascular beds. The adhesion complex was blocked by administering the anti-CD18 mAb 60.3. The data show that mAb 60.3 blocks PMN emigration into inflammatory foci in the abdominal wall produced by implanting sponges containing either hydrochloric acid, Streptococcus pneumoniae, Escherichia coli endotoxin, or PMA. mAb 60.3 also inhibited PMN emigration in response to peritoneal instillation of S. pneumoniae. The effect of mAb 60.3 on PMN emigration in the lungs varied depending upon the stimulus. PMN failed to migrate into the PMA-induced pneumonia; however, mAb 60.3 pretreatment only partially inhibited endotoxin-induced pneumonia and did not inhibit S. pneumoniae or hydrochloric acid-induced pneumonias. PMN lavaged from the alveolar spaces in the Streptococcal pneumonia had similar quantities of mAb 60.3 bound to their surfaces as the circulating PMN. We conclude that the CD11/CD18 complex mediates PMN adherence in the systemic circulation. However, PMN adherence in the pulmonary circulation may occur by either CD18-dependent or -independent mechanisms that are specific to the inciting stimulus.

High endothelial-like venules in chronically inflamed periodontal tissues exchange polymorphs.

A survey of 58 gingival biopsies revealed the presence of periodontal high endothelial-like venules (PHELVs) in chronically inflamed gingival tissues. PHELVs were found to exchange polymorphonuclear cells (PMNs) almost exclusively in advanced periodontitis, with PMNs greatly exceeding the number of mononuclear cells found in PHELVs (P less than 0.001). Electron microscopy confirmed the emigration of PMNs from these vessels. The enzyme histochemical and ultrastructural features as well as the 35SO4 uptake properties of PHELVs were similar to those of the well-characterized high endothelial venules (HEVs) of rat lymph nodes. It is generally accepted that HEVs in lymphoid tissues and inflammatory sites are specially adapted to assist in the emigration of lymphocytes. However, the observation of preferential PMN emigration in the apparent absence of lymphocyte exchange from PHELVs compels further investigation of other possible functions for HEVs. In relation to this, endothelial cells are capable of producing potent cytokines and inflammatory mediators which may contribute to the development of lesions, and the possibility is discussed that high endothelial cells are functionally adapted to enhance the production of such factors.

Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules.

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.