Effects of polysaccharide fucoidin on cerebrospinal fluid interleukin-1 and tumor necrosis factor alpha in pneumococcal meningitis in the rabbit

The inflammatory response in bacterial meningitis is mediated by cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1), which are produced in the subarachnoid space by different cells, e.g., leukocytes, astrocytes, and microglia. The recruitment of leukocytes into the cerebrospinal fluid (CSF) has been shown to contribute to the neurological damage in this disease, a process which could be enhanced by treatment with antibiotics. In this study, we have used a rabbit meningitis model for two sets of experiments with intracisternal (i.c.) injections of Streptococcus pneumoniae. First, pneumococcal cell wall (PCW) components were injected i.c., inducing an inflammatory response with pleocytosis and increased levels of CSF TNF-alpha) and IL-1 at 6 and 12 h after PCW injection. Treatment with fucoidin, known to inhibit leukocyte rolling, abolished pleocytosis and inhibited the release of TNF-alpha and IL-1. In the second experiment, live pneumococcal bacteria were injected i.c. and treatment with one dose of ampicillin (40 mg/kg of body weight intravenously) was given 16 h after induction of meningitis, causing a sevenfold increase in CSF leukocytes over a 4-h period. CSF IL-1 levels at 16 h were high but did not increase further at 20 h. Also, CSF TNF-alpha levels were high at 16 h and tended to increase at 20 h. Fucoidin treatment prevented the antibiotic-induced increase of CSF leukocytes but had no effect on the TNF-alpha and IL-1 levels. Taken together, fucoidin reduced CSF TNF-alpha and IL-1 levels in acute bacterial meningitis induced by PCW fragments but had no effect later in the course of the disease, when live bacteria were used and an inflammatory increase was caused by a dose of antibiotics.     

The Polysaccharide Fucoidin Inhibits the Antibiotic-Induced Inflammatory Cascade in Experimental Pneumococcal Meningitis

There is evidence that the treatment of bacterial meningitis with antibiotics liberates harmful bacterial products in the subarachnoid space (SAS). This enhances meningeal inflammation and in particular the recruitment of leukocytes into the cerebrospinal fluid (CSF), which has been shown to be more harmful than beneficial in this disease. In this study, we used a rabbit meningitis model based on intracisternal injection of live Streptococcus pneumoniae. Ampicillin (40 mg/kg of body weight given intravenously [i.v.] 16 h after induction of meningitis) caused a fivefold increase in CSF leukocytes over a 4-h period. Inhibition of leukocyte rolling by treatment with the polysaccharide fucoidin (10 mg/kg, i.v.) prevented the enhanced leukocyte extravasation into the SAS and attenuated the leakage of plasma proteins over the blood-brain barrier. These results suggest that certain polysaccharides that block leukocyte rolling have the potential to reduce leukocyte-dependent central nervous system damage in bacterial meningitis.     

Rabbit leukocyte adhesion molecules CD11/CD18 and their participation in acute and delayed inflammatory responses and leukocyte distribution in vivo

In humans the glycoprotein complexes CD11/CD18 mediate leukocyte adhesion to cells. Mouse monoclonal antibodies (mAb) 60.3, 7E4, and IB4 to human CD18, found to cross-react with rabbit white blood cells, were used to identify the antigen in rabbit cells and to study adherence of rabbit leukocytes in vitro and in vivo. These antibodies labeled almost all unfractionated rabbit blood leukocytes and immunoprecipitated surface glycopolypeptides with apparent molecular weights of 85,000 and 150,000 from these cells. Adhesion of purified rabbit polymorphonuclear cells (PMNs) to cultured vascular endothelial cells in the presence of phorbol ester was blocked by the antibodies in a dose-dependent manner. The acute inflammatory response characterized by local accumulation of PMNs and concomitant plasma extravasation following intradermal injections of zymosan-activated serum (ZAS) in rabbits was inhibited in animals pretreated intravenously with anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 mAb. Intravital microscopy of the rabbit tenuissimus muscle demonstrated that anti-CD18 treatment specifically blocked the adhesion of activated leukocytes to the venular endothelium and thereby the subsequent diapedesis of these cells into the extravascular space. The lymphocyte-dependent tissue swelling resulting from a delayed-type hypersensitivity reaction in the rabbit ear was partially inhibited by anti-CD18 mAb. Systemic anti-CD18 treatment induced a pronounced increase in the number of circulating mononuclear and polymorphonuclear cells with a maximum at 24 hr after injection of the antibody. It is concluded that GP150/GP85 is the rabbit homologue of human CD11/CD18, and that leukocyte-cell adhesion mediated by these glycoprotein complexes participates in acute and delayed inflammatory responses and leukocyte distribution in vivo.     

Tumor necrosis factor and interferon-gamma induce distinct patterns of endothelial activation and associated leukocyte accumulation in skin of Papio anubis.

Recombinant human interferon (IFN)-gamma (2 X 10(4) or 2 X 10(5) U), tumor necrosis factor (TNF, 10(4) or 10(5) U), or both were injected intracutaneously into baboons (Papio anubis), and biopsies were examined at various intervals for evidence of altered endothelial cell antigen expression, endothelial morphology, and leukocyte infiltration. IFN-gamma induced increased binding of anti-HLA-DP mAb by 24 hours and a mild-to-moderate accumulation of mononuclear cells. TNF induced increased binding of anti-endothelial leukocyte adhesion molecule (ELAM)-1 mAb by 2 hours that was associated with polymorphonuclear leukocyte accumulation, and increased binding of anti-intercellular adhesion molecule (ICAM)-1 mAb by 9 hours that was associated with the onset of progressive mononuclear leukocyte accumulation. TNF also caused endothelial cell hypertrophy and increased vascular permeability. The combination of IFN-gamma and TNF induced a set of changes that qualitatively resemble those of a delayed hypersensitivity reaction to simian agent 8 envelope antigen. These findings are consistent with the concept that cytokine-activated endothelium plays an important role in the adhesion and subsequent extravasation of leukocytes during immune inflammation.     

Important role of CD18 in TNF-<Emphasis Type="Italic">α</Emphasis>-induced leukocyte adhesion in muscle and skin venules in vivo

OBJECTIVE: To examine the role of CD 18 in tumor necrosis factor-alpha (TNF-alpha)-induced leukocyte adhesion and extravasation in vivo. MATERIAL: Male wild-type (WT) and mutated mice with hypomorphic expression of CD 18. METHODS: Intravital microscopy was used to quantitate leukocyte-endothelium interactions provoked by TNF-alpha (0.5 microg) in the cremaster muscle and dorsal skin microcirculation. Tissue recruitment of leukocytes was evaluated in wholemounts of the cremaster muscle and in air pouches in the dorsal skin after TNF-alpha stimulation. RESULTS: TNF-alpha markedly increased venular leukocyte adhesion and recruitment in the cremaster muscle and skin in WT. Notably, in CD 18-targeted animals, leukocyte adhesion triggered by TNF-alpha challenge was significantly reduced by 58% and 72% in venules of the cremaster muscle and skin, respectively. Moreover, in CD18-mutants, tissue accumulation of polymorphonuclear leukocytes (PMNLs) provoked by TNF-alpha in the muscle and skin was decreased by 84% and 70%, respectively. Interestingly, the observed level of reduction in TNF-alpha-induced neutrophil adhesion and recruitment in CD18 gene-targeted animals corresponded well with the decrease in CD 18 expression on neutrophils from these mice, i.e. the surface density of CD18 was reduced by 77% in mutants compared to WT. Differential analysis revealed that the extravascular leukocytes comprised more than 90% PMNLs, indicating that neutrophils were the main inflammatory cell responding to TNF-alpha activation. Notably, the expression of CD18 increased by more than two-fold on extravasated neutrophils compared to circulating neutrophils in the peripheral blood both in WT and mutant animals. CONCLUSIONS: These findings suggest that CD18 is a dominant mediator of firm neutrophil adhesion to venular endothelial cells in the muscle and skin stimulated by TNF-alpha in vivo. In addition, this decreased adhesion in CD18-mutants attenuates leukocyte extravasation in response to TNF-alpha activation. Thus, inhibition of CD 18-function may provide an important strategy to inhibit leukocyte recruitment in cytokine-dependent diseases.     

Recognition of pneumococcal peptidoglycan: an expanded,pivotal role for LPS binding protein

Lipopolysaccharide binding protein (LBP) has a well-established role in LPS-induced immune responses. Here, we report that LBP also plays an essential role in the innate immune response to Gram-positive pneumococci, specifically to their major inflammatory component, pneumococcal cell wall (PCW). LBP was present in the CSF of patients with meningitis, and LBP-deficient mice failed to develop meningeal inflammation. LBP enhanced PCW-induced cell signaling and TNF-alpha release. LBP bound specifically to PCW multimers, indicating novel lipid-independent binding capability for LBP. We propose the iterative anionic groups along the glycan backbone of the cell wall are a crucial structure for recognition by LBP. Such a function for LBP expands its role to Gram-positive infections.     

Peripheral injection of lipopolysaccharide prevents brain recruitment of leukocytes induced by central injection of interleukin-1

I.c.v. injection of interleukin-1beta induces infiltration of leukocytes into the brain. I.p. injection of bacterial endotoxin lipopolysaccharide induces the expression of interleukin-1 in the CNS without causing the entry of leukocytes into the brain. This suggests that during systemic inflammation trafficking of potentially damaging leukocytes into the CNS is inhibited. In this study, we investigated the effects of peripheral injection of lipopolysaccharide on brain leukocyte recruitment induced by i.c.v.-interleukin-1 in mice. I.c.v.-interleukin-1 induced widespread infiltration of leukocytes into the brain 16 h after the injection. Pretreatment with i.p.-lipopolysaccharide 2 h before the i.c.v. interleukin-1 injection completely blocked interleukin-1-induced leukocyte infiltration, whereas i.p.-LPS only attenuated the effect of interleukin-1 if it was given 12 h before i.c.v. interleukin-1 injection. I.p.-lipopolysaccharide given 24 h before i.c.v. interleukin-1 injection did not alter interleukin-1 induced leukocyte infiltration. I.c.v.-interleukin-1 induced expression of p- and e-selectins in brain vasculatures prior to the appearance of leukocytes in the brain parenchyma. Induction of p- and e-selectin was inhibited by the pretreatment of i.p.-lipopolysaccharide 2 h, but not 24 h, before i.c.v.-interleukin-1 injection. I.c.v.-interleukin-1-induced leukocyte infiltration was diminished in both e- and p- selectin knockout animals. These results suggest that systemic inflammation actively inhibits recruitment of leukocytes by CNS. Inhibition of the expression of p- and e-selectins is a mechanism by which peripheral inflammation regulate CNS leukocyte recruitment.     

A three-dimensional model system to study the interactions between human leukocytes and endothelial cells

Leukocyte adhesion to endothelial cells and migration into the subendothelial matrix was studied with a three-dimensional model system, consisting of human endothelial cells cultured on a loose collagen matrix. We developed a new method to separate the endothelial cell monolayer and adhering leukocytes, from the subendothelial matrix, allowing simultaneous analysis of leukocyte adhesion and transendothelial migration. Monocytes adhered more avidly to untreated endothelial cells than did neutrophils (2.5 +/- 0.3 vs. 1.0 +/- 0.2 leukocytes per endothelial cell). Only a small fraction (10%-20%) of these leukocytes migrated into the subendothelium. Pretreatment of endothelial cells with interleukin 1 (IL 1) enhanced adhesion (20%), but not migration of monocytes. In contrast, neutrophil adhesion was markedly and in a time-dependent manner increased by IL 1 treatment (i.e. 200% after 6 h and 110% after 24 h of IL 1 treatment). Moreover, IL 1 pretreatment enhanced neutrophil migration twofold. Activation of leukocytes with formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced both monocyte and neutrophil adhesion, but did not affect leukocyte migration. Under all conditions, monocyte adhesion was only partly (30%-40%) inhibited by monoclonal antibodies (mAb) against the common beta subunit of the leukocyte-cell adhesion molecules (LeuCAM: CD18) and 25%-30% by mAb against the alpha subunit of LFA-1 (CD11a). In contrast, mAb against the alpha subunits of Mac-1 (CD11b) and p150.95 (CD11c) were hardly effective. fMLP-mediated neutrophil adhesion was reduced to below baseline levels by anti-LeuCAM (CD18) mAb, whereas the LeuCAM contribution in IL 1-mediated neutrophil adhesion was less pronounced and varied in time. IL 1-mediated neutrophil migration, however, was completely blocked by anti-LeuCAM mAb. fMLP-mediated neutrophil adhesion was inhibited by mAb against the alpha subunits of Mac, while mAb against the alpha subunits of LFA-1 and Mac-1 both reduced IL 1-mediated adherence. In summary, we describe a novel leukocyte adhesion/migration method and demonstrate that the contribution of the LeuCAM complex in leukocyte-endothelium interaction varies depending on cell type and stimulus used.     

The Effects of IB4, a Monoclonal Antibody to the CD18 Leukocyte Integrin on Phorbol Myristate Acetate (PMA)-Induced Polymorphonuclear Leukocyte (PMN) Accumulation and Endothelial Injury in Rabbit Lungs

A model of acute lung injury induced by intravenous phorbol myristate acetate (PMA) is described. The model is characterized by the accumulation of polymorphonuclear leukocytes (PMNs) and a hemorrhagic edema in bronchoalveolar lavage (BAL) fluid when measured 6 h following the administration of PMA (60 g/kg, i.v.). It was also determined that PMA induces acute leukopenia and neutropenia which were maximal at 5 min following the injection of PMA and were sustained for at least 6 h, with circulating leukocyte numbers returning to control values by 24 h. The extents to which the inflammatory and systemic changes induced by PMA were dependent on the surface expression on leukocytes of the 2-integrins was assessed by comparing responses to PMA in control animals and animals pretreated with the anti-CD18 monoclonal antibody IB4. The administration of IB4 (1 mg/kg, i.v.) 15 min before PMA did not alter the time course or extent of PMA-induced leukopenia and neutropenia. In contrast IB4 administration (0.1 to 1 mg/kg) produced a dose dependent inhibition of PMN accumulation and plasma extravasation measured in BAL fluid. IB4 (1 mg/kg) completely inhibited PMA evoked increases in plasma extravasation (94.5 ± 1.7%, N = 4) and hemorrhage (95.2 ± 2.1%, N = 4) whereas PMN accumulation in BAL fluid was inhibited by 77.8 ± 3.8% (mean ± SEM, N = 4). Thus, a small, but reproducible, component of the PMA-induced PMN accumulation was not inhibited using this regimen of IB4 administration. If IB4 administration was delayed for 3 h post injection of PMA and bronchoalveolar lavage performed 3 h later, the extents of PMN accumulation and edema formation were similar to those observed 3 h following PMA challenge in control animals not dosed with IB4. This suggests that administration of IB4 during an ongoing inflammatory response is capable of preventing the further development of inflammatory changes and further supports the therapeutic potential of CD18 blockade in conditions such as adult respiratory distress syndrome.     

The role of E- and P-selectin in neutrophil and monocyte migration in adjuvant-induced arthritis in the rat

The role of the endothelial adhesion molecules E- and P-selectin in leukocyte accumulation in arthritis is not known. We investigated this role in rat adjuvant arthritis by employing adhesion function-blocking monoclonal antibodies (mAb) to rat P- and E-selectin. The acute migration (2 h) of radiolabeled rat blood neutrophils and monocytes to joints and skin was determined. Anti-P-selectin mAb significantly reduced accumulation of monocytes (by 50%) and neutrophils (by 40%) in the talar joint, and of neutrophils in tail joints (by 90%). Anti-E-selectin mAb alone did not attenuate leukocyte migration, but when combined with anti-P-selectin mAb, it enhanced inhibition of neutrophil accumulation in the talar and carpal joints. In the same animals, anti-P-selectin mAb significantly inhibited neutrophil and monocyte migration to dermal inflammatory reactions induced by zymosan-activated rat serum (ZAS) containing the chemotactic factor C5a_{des Arg}, endotoxin (LPS), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In contrast, anti-E-selectin mAb alone had no effect on monocyte or neutrophil accumulation in inflamed skin of arthritic animals, but again enhanced the inhibition when combined with mAb to P-selectin. The addition of anti-L-selectin mAb to anti-P- and E-selectin mAb did not further suppress monocyte or neutrophil migration to inflamed skin or joints. These results demonstrate that optimal leukocyte migration to arthritic joints and inflamed skin is P-selectin dependent, and E-selectin is not essential. However, E-selectin contributes to migration when P-selectin mechanisms are not operative. L-selectin does not play a role in E- and P-selectin-independent leukocyte migration to joints or skin inflammation in arthritic rats. However, it is likely that additional selectin-independent pathways also mediate neutrophil and monocyte migration to joint and skin inflammation.     

Inhibition of leukocyte entry into the brain by the selectin blocker fucoidin decreases interleukin-1 (IL-1) levels but increases IL-8 levels in cerebrospinal fluid during experimental pneumococcal meningitis in rabbits

The polysaccharide fucoidin is a selectin blocker that inhibits leukocyte recruitment into the cerebrospinal fluid (CSF) during experimental pneumococcal meningitis. In the present study, the effect of fucoidin treatment on the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-8 into the CSF was investigated. Rabbits (n = 7) were treated intravenously with 10 mg of fucoidin/kg of body weight every second hour starting 4 h after intracisternal inoculation of approximately 10(6) CFU of Streptococcus pneumoniae type 3 (untreated control group, n = 7). CSF samples were obtained every second hour during a 16-h study period. Treatment with fucoidin caused a consistent and significant decrease in CSF IL-1 levels (in picograms per milliliter) between 12 and 16 h (0 versus 170, 0 versus 526, and 60 versus 1,467, respectively; P < 0.02). A less consistent decrease in CSF TNF-alpha levels was observed in the fucoidin-treated group, but with no significant difference between the two groups (P > 0.05). In contrast, there was no attenuation in CSF IL-8 levels. Indeed, there was a significant increase in CSF IL-8 levels (in picograms per milliliter) in the fucoidin-treated group at 10 and 12 h (921 versus 574 and 1,397 versus 569, respectively; P < 0.09). In conclusion, our results suggest that blood-derived leukocytes mainly are responsible for the release of IL-1 and to some degree TNF-alpha into the CSF during pneumococcal meningitis, whereas IL-8 may be produced by local cells within the brain.     

Linomide abolishes leukocyte adhesion and extravascular recruitment induced by tumor necrosis factor alpha in vivo

The immunomodulator Linomide (roquinimex) ameliorates the development of numerous inflammatory and immunological diseases, including sepsis, arthritis, and encephalomyelitis. However, the mechanism underlying this protective effect of Linomide remains unclear. In this study, we wanted to evaluate the effect of Linomide treatment on the different steps in the extravasation process of leukocytes stimulated by tumor necrosis factor alpha (TNF-alpha) in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle microcirculation. We found that pretreatment with Linomide dose-dependently (3-300 mg/kg) reduced TNF-alpha-induced leukocyte adhesion and tissue recruitment. Notably, at 300 mg/kg response to TNF-alpha was nearly abolished, i.e. leukocyte adhesion was decreased by 83% and recruitment by 86%. In fact, the anti-inflammatory effect of this dose of Linomide corresponded in magnitude to the potency of 10 mg/kg of dexamethasone. Moreover, administration of Linomide did not alter the systemic leukocyte counts. On the other hand, 1-10 mg/kg of dexamethasone decreased the circulating number of mononuclear leukocytes by 77%. Taken together, our novel findings demonstrate that Linomide is a potent inhibitor of leukocyte adhesion and recruitment in cytokine-activated tissues. These data may help explain the documented protection provided by Linomide in inflammatory diseases characterized by cytokine and leukocyte accumulation.     

Diagnostic Accuracy of Presepsin (sCD14-ST) for Prediction of Bacterial Infection in Cerebrospinal Fluid Samples from Children with Suspected Bacterial Meningitis or Ventriculitis

Children with temporary external ventricular drains (EVD) are prone to nosocomial infections. Diagnosis of bacterial meningitis and ventriculitis in these children is challenging due to frequent blood contamination of cerebrospinal fluid (CSF) and the presence of chemical ventriculitis. The aim of this study was to compare diagnostic accuracy of presepsin (sCD14-ST), a novel biomarker of bacterial infection in CSF, to predict bacterial infection in comparison to the accuracy of established biomarkers like those demonstrated in biochemical analysis of CSF. We conducted a prospective study with 18 children with suspected bacterial meningitis or ventriculitis who had 66 episodes of disease. CSF samples were taken from external ventricular drainage. We measured presepsin in CSF, as well as CSF leukocyte count, glucose, and proteins. CSF was also taken to prove bacterial infection with culture methods or with 16S rRNA gene broad-range PCR (SepsiTest; Molzym, Germany). Infection was clinically confirmed in 57 (86%) episodes of suspected meningitis or ventriculitis. Chemical ventriculitis was diagnosed in 9 (14%) episodes of suspected meningitis or ventriculitis. Diagnostic accuracies presented as area under the curve (AUC) for sCD14-ST, leukocytes, and proteins measured in CSF were 0.877 (95% confidence interval [CI], 0.793 to 0.961), 0.798 (95% CI, 0.677 to 0.920), and 0.857 (95% CI, 0.749 to 0.964), respectively. With CSF culture, we detected bacteria in 17 samples, compared to 37 detected with broad-range PCR. It was found that presepsin was present at a significantly higher level in children with clinically proven ventriculitis than in those without meningitis or ventriculitis. Diagnostic accuracies of presepsin were superior to those of leukocytes or proteins in CSF. Presepsin-guided 16S rRNA gene PCR could be used in everyday clinical practice to improve etiological diagnosis of meningitis and ventriculitis and to prescribe more appropriate antibiotics.     

The role of complement in inflammation during experimental pneumococcal meningitis

The mechanism whereby an effective bactericidal inflammatory reaction develops in the subarachnoid space is not clearly defined. While normal cerebrospinal fluid is deficient in complement, immunoglobulin and leukocytes, these serum components appear in cerebrospinal fluid (CSF) during the course of bacterial meningitis. Using a rabbit model of pneumococcal meningitis we examined the role of the alternate complement pathway in three early events important to the defense of the subarachnoid space: leukocyte chemotaxis, phagocyte mediated bacterial killing, and clearance of bacterial components from the cerebrospinal fluid space. Rabbits treated with cobra venom factor to deplete complement were inoculated intracisternally with encapsulated (type II or XIX) pneumococci. Following complement depletion, there was a dramatic (at least 100-fold) decrease in the LD50 for these strains. Nevertheless, complement depletion did not affect the magnitude of CSF leucocytosis or the rate of clearance of bacterial particles from CSF. A short delay in the appearance of leukocytes in CSF was found in the absence of complement. The major effect of complement depletion, however, was to diminish the efficiency of leukocyte mediated killing of encapsulated bacteria in the CSF. Although the short delay in the onset of leukocytosis in the complement depleted animals is consistent with a chemotactic role of complement in the normal animal, the quantitatively normal leukocytosis in the complement depleted rabbits clearly indicates that important chemotaxins other than complement function in CSF. Inhibition of leukocytosis by indomethacin and diclofenac suggests that metabolite(s) of the arachidonic acid pathway may perform such a chemotactic role. A major role of complement in the defense of the subarachnoid space appears to be as an opsonin needed for the effective bactericidal activity of leukocytes. It is the lack of this function that best explains the greatly decreased LD50 value of encapsulated pneumococci in the complement depleted animal.     

L-Selectin Shear Thresholding Modulates Leukocyte Secondary Capture

Transient homotypic adhesions between flowing leukocytes and those previously adherent on the vessel wall has been proposed to amplify the accumulation of leukocytes at sites of inflammation. While adhesion of leukocytes to the vessel wall (primary capture) is mediated primarily by P-selectin on the endothelium and P-selectin Glycoprotein Ligand-1 (PSGL-1) on the leukocyte, the homotypic interactions leading to downstream leukocyte adhesion (secondary capture) are mediated primarily by reciprocal interactions between PSGL-1 and L-selectin on apposing leukocytes. One consequence of leukocyte secondary capture events are the formation of strings of adherent leukocytes as each recently captured leukocyte in turn captures another one flowing over its surface. Interestingly, PSGL-1-L-selectin interactions also mediate leukocyte hydrodynamic shear thresholding, whereby leukocyte rolling on purified L-selectin ligands such as PSGL-1 is maximized at a wall shear stress of ∼1 dyne/cm² and minimized at both higher and lower flow rates. Using a novel quantitative method, we analyzed leukocyte string formation in vitro and found that hydrodynamic shear thresholding precluded secondary capture at low shear stresses yet amplified it at high shear stresses. Addition of the L-selectin mAb DREG-56 strongly inhibited leukocyte string formation, suggesting adhesion contributed significantly to hydrodynamic interactions in secondary capture processes. Taken together, the data suggest that secondary capture is modulated by the shear thresholding property of L-selectin. L-selectin mediated shear thresholding may therefore play a significant role in the regulation of leukocyte secondary capture in addition to recently described hydrodynamic recruitment mechanisms.     

Systemic neutralization of interleukin-8 markedly reduces neutrophilic pleocytosis during experimental lipopolysaccharide-induced meningitis in rabbits

Interleukin-8 (IL-8) is elevated in the cerebrospinal fluid (CSF) of patients with meningitis and is proposed to participate in subarachnoid-space pleocytosis. However, intracisternal injection of IL-8 into rabbits failed to induce indices typical of meningitis (leukocyte, tumor necrosis factor, or protein accumulation in the CSF or histopathological changes), indicating that merely increasing the CSF level of this chemokine is insufficient to induce inflammation in this anatomical site. IL-8 treatment did not affect inflammatory responses to subsequently intracisternally administered lipopolysaccharide (LPS). IL-8 was chemotactic for rabbit neutrophils in vitro, and subcutaneous injection of IL-8 (diluted in buffer or CSF) proved the in vivo activity of this peptide and suggested the absence of an IL-8 inhibitor in normal rabbit CSF. LPS-dependent pleocytosis was only slightly diminished by intracisternally administered murine anti-rabbit IL-8 monoclonal antibody (MAb) WS-4 but was dramatically reduced by intravenously administered MAb. Therefore, elevated CSF IL-8 levels may contribute to, but cannot solely account for, neutrophil influx into the subarachnoid space during meningitis. However, inhibition of IL-8 activity of the bloodstream side of the blood-brain barrier effectively reduces pleocytosis, indicating a central role of IL-8 in neutrophil influx into CSF during bacterial meningitis. Thus, inhibition of IL-8 is a possible therapeutic target for adjunct treatment of meningitis.     

HECA-452 is a non-function blocking antibody for isolated sialyl Lewis x adhesion to endothelial expressed E-selectin under flow conditions

E-selectin, expressed on inflamed endothelium, and sialyl Lewis x (sLe(x)), present on the surface of leukocytes, play a key role in leukocyte-endothelial interactions during leukocyte recruitment to sites of inflammation. HECA-452 is a monoclonal antibody (mAb) that recognizes sLe(x) and is routinely used by investigators from diverse fields who seek to unravel the mechanisms of leukocyte adhesion. The data regarding the ability of HECA-452 to inhibit carbohydrate-mediated leukocyte adhesion to E-selectin remains conflicted, in part due to the presence of a variety of potential E-selectin reactive moieties on leukocytes. Recognizing this, we utilized a complementary approach to gain insight into HECA-452 adhesion assays. Specifically, we used sLe(x) microspheres to investigate the hypothesis that HECA-452 is a non-function blocking mAb for isolated sLe(x) mediated adhesion to endothelial expressed E-selectin. Flow cytometric analysis revealed that HECA-452 recognizes and binds to the sLe(x) microspheres. Perfusion of the sLe(x) microspheres over human umbilical vein endothelial cells (HUVEC) at 1.5dyn/cm(2) revealed that the microspheres attach to 4h interleukin (IL)-1β activated HUVEC specifically via E-selectin. Pretreatment of the sLe(x) microspheres with HECA-452 did not influence sLe(x) microsphere initial tethering and accumulation on IL-1β activated HUVEC. Neuraminidase and fucosidase treatments of sLe(x) microspheres revealed that sialic acid and fucose are required for E-selectin binding, whereas HECA-452 recognition of sLe(x) does not depend on the fucose moiety to the extent required for E-selectin recognition. This latter finding suggests there are potential subtle differences between the sLe(x) antigens for E-selectin and HECA-452. Combined, the data indicate that HECA-452 is a non-inhibitor of sLe(x)-mediated adhesion to endothelial expressed E-selectin.     

P-selectin requirement for neutrophil accumulation and injury in the direct passive Arthus reaction

The aim of this study was to investigate the role of P-selectin in the accumulation of neutrophils in the direct passive Arthus reaction in rat skin. Direct passive Arthus dermal reaction was induced in male Sprague-Dawley (SD) rats by a single i.v. injection of rat anti-sheep globulin (SG) 1 h before i.d. injection of SG antigen. Anti-P-selectin or irrelevant control antibody was given 1 h before rat anti-SG injection. Complement depletion was also performed in a separate group by pretreatment with cobra venom factor (CVF). In all groups dermal swelling was assessed 4 h after antigen challenge. Four hours after antigen challenge, rats treated with control antibody developed skin swelling (2.29 ± 0.47 mm), prominent complement deposition and neutrophil accumulation. This response was associated with local up-regulation of endothelial P-selectin. Pre-treatment with anti-P-selectin antibody 1 h before passive Arthus induction prevented skin swelling (0.29 ± 0.06 mm, P < 0.05, cf with control antibody treatment), neutrophil accumulation and up-regulation of endothelial P-selectin despite complement deposition. CVF treatment prevented complement deposition, neutrophil accumulation and skin swelling (0.13 ± 0.07 mm, P < 0.05, cf with saline treatment). However, endothelial P-selectin expression was still present. Inhibition of skin swelling and neutrophil accumulation in direct passive Arthus by functional inhibition of P-selectin suggest a pivotal role for this adhesion molecule in this inflammatory process. These results also suggest that multiple steps are involved in the evolution of direct passive Arthus, including both P-selectin expression and complement activation. However, while complement activation is essential for neutrophil accumulation and expression of dermal injury, P-selectin up-regulation initiated by antibody/antigen deposition occurs independently of complement activation.     

Intravenous Granulocyte Colony-Stimulating Factor Increases the Release of Tumour Necrosis Factor and Interleukin-1β into the Cerebrospinal Fluid, But Does Not Inhibit the Growth of Streptococcus pneumoniae in Experimental Meningitis

Granulocyte colony-stimulating factor (G-CSF) possesses an antimicrobial effect in several animal models of infection. To evaluate a possible effect of G-CSF on the course of pneumococcal meningitis, rabbits infected intracisternally (i.c.) with Streptococcus pneumoniae type 3 (n = 7) received 50 microgram/kg of rhG-CSF intravenously (i.v.) 1 h prior to infection. Seven infected animals served as controls. Uninfected rabbits received 10 microgram of G-CSF (n = 3), 2 microgram G-CSF (n = 3) or saline (n = 3) i.c. G-CSF injected i.c. did not produce cerebrospinal fluid (CSF) leucocytosis. Compared with the control group, i.v. G-CSF given prior to i.c. infection increased the percentage of granulocytes in blood 6 h and 12 h after infection. Twelve hours after infection, CSF tumour necrosis factor (TNF) activity and CSF interleukin (IL)-1beta concentrations were significantly higher in G-CSF-treated animals. G-CSF did not decrease bacterial growth in the subarachnoid space and the CSF leucocyte densities were not influenced. At 24 h after infection, G-CSF did not reduce the CSF concentrations of neurone-specific enolase and the density of apoptotic neurones in the dentate gyrus of the hippocampus. In conclusion, i.v. G-CSF increased the concentration of pro-inflammatory cytokines in the CSF but did not decrease the growth of Streptococcus pneumoniae in the subarachnoid space.     

The protein tyrosine kinase inhibitor AG126 prevents the massive microglial cytokine induction by pneumococcal cell walls.

Central nervous system (CNS) infections caused by Streptococcus pneumoniae still have a disastrous outcome. Underlying immunological and CNS cellular events are largely enigmatic. We used pneumococcal cells walls (PCW) to investigate microglial responses as these cells are prominent sensors and effectors during neuropathological changes. PCW stimulation of mouse microglia in vitro evoked the release of the cyto- and chemokines, TNF-alpha, IL-6, IL-12, KC, MCP-1, MIP-1alpha, MIP-2 and RANTES as well as soluble TNF receptor II, a potential TNF-alpha antagonist. The release induction followed extremely steep dose-response relations, and short exposure periods (15 min) were already sufficient to trigger substantial responses. PCW signaling controlling the release depended on both p38 and p42/p44 (ERK2/ERK1) MAP kinase activities. The kinase inhibitor, tyrphostin AG126 prevented the PCW-inducible phosphorylation of p42/p44(MAPK), potently blocked cytokine release and drastically reduced the bioavailable TNF-alpha, since it only marginally affected the release of soluble TNF receptors. Moreover, in an in vivo model of pneumococcal meningitis, AG126 significantly attenuated the PCW-induced leukocyte influx to the cerebrospinal fluid. The findings imply that pneumococcal CNS infection can cause a rapid and massive microglial activation and that ERK/MAPK pathway(s) are potential targets for pharmacological interventions.