Sialic acid residues play a pivotal role in alpha(1)-acid glycoprotein (AGP)-induced generation of reactive oxygen species in chemotactic peptide pre-activated neutrophil granulocytes.

OBJECTIVE: We have recently shown that terminal sialic acid residues are essential for alpha(1)-acid glycoprotein (AGP)-induced Ca(2+) mobilization in neutrophils. The aim of the present study was to establish the importance of sialic acid residues on AGP in modulating human neutrophil functions, with emphasis on the generation of reactive oxygen species (ROS). MATERIALS AND METHODS: ROS were measured by luminol-enhanced chemiluminescence in isolated human neutrophils. RESULTS: We found that AGP did not provoke ROS generation in resting or L-selectin presensitized neutrophils. Moreover, AGP did not affect the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS generation, but it slightly suppressed opsonized zymosan-induced responses. However, when the neutrophils were prestimulated with fMLP, the subsequent addition of AGP provoked a marked ROS response. Dose-response studies and time studies revealed that the ROS generating capacity of AGP was highest at a concentration of 0.05 mg/ml and when given 3-10 min after addition of fMLP. A desialylated form of AGP or pretreatment of neutrophils with 3'- and 6'-sialyllactose caused a substantially lower ROS response in neutrophils prestimulated with fMLP. CONCLUSIONS: Our data show that AGP can stimulate a second ROS response in fMLP preactivated neutrophils and that terminal sialic acid residues on AGP play a crucial role in this regard.

Role of anti-L-selectin antibody in burn and smoke inhalation injury in sheep.

We hypothesized that the antibody neutralization of L-selectin would decrease the pulmonary abnormalities characteristic of burn and smoke inhalation injury. Three groups of sheep (n = 18) were prepared and randomized: the LAM-(1-3) group (n = 6) was injected intravenously with 1 mg/kg of leukocyte adhesion molecule (LAM)-(1-3) (mouse monoclonal antibody against L-selectin) 1 h after the injury, the control group (n = 6) was not injured or treated, and the nontreatment group (n = 6) was injured but not treated. All animals were mechanically ventilated during the 48-h experimental period. The ratio of arterial PO2 to inspired O2 fraction decreased in the LAM-(1-3) and nontreatment groups. Lung lymph flow and pulmonary microvascular permeability were elevated after injury. This elevation was significantly reduced when LAM-(1-3) was administered 1 h after injury. Nitrate/nitrite (NO(x)) amounts in plasma and lung lymph increased significantly after the combined injury. These changes were attenuated by posttreatment with LAM-(1-3). These results suggest that the changes in pulmonary transvascular fluid flux result from injury of lung endothelium by polymorphonuclear leukocytes. In conclusion, posttreatment with the antibody for L-selectin improved lung lymph flow and permeability index. L-selectin appears to be principally involved in the increased pulmonary transvascular fluid flux observed with burn/smoke insult. L-selectin may be a useful target in the treatment of acute lung injury after burn and smoke inhalation.

Soluble L-selectin attenuates tumor necrosis factor-alpha-mediated leukocyte adherence and vascular permeability: a protective role for elevated soluble L-selectin in sepsis.

OBJECTIVE: We have previously demonstrated that leukocyte delivery to remote sites is decreased in sepsis and that increased concentrations of soluble L-selectin are, in part, responsible for this finding. Given that leukocytes have been implicated in the pathogenesis of vascular leakage, we hypothesized that the elevated soluble L-selectin concentrations in sepsis may translate into decreased inflammation-mediated leukocyte-endothelial cell interactions and vascular leakage at these sites. DESIGN: Prospective, controlled animal study. SETTING: Surgical research laboratory in a university hospital. SUBJECTS: Swiss white male mice weighing 25-35 g. INTERVENTIONS: Mice were randomized to one of three study groups: intracremaster tumor necrosis factor-alpha with subsequent intravenous bicarbonate buffered solution; intracremaster tumor necrosis factor-alpha with intravenous soluble L-selectin (10 microg/mL); and intracremaster bicarbonate buffered solution with intravenous bicarbonate buffered solution. The cremaster muscle was prepared for both light and fluorescence intravital microscopy 2 hrs after intracremaster injection, and fluorescein isothiocyanate-labeled albumin was injected intravenously. Leukocyte-endothelial interactions (rolling flux, rolling velocity, and adherence) were counted off-line. Postcapillary venule leakage was determined by the permeability index (perivenular/intravenular fluorescence) after intravenous injection of fluorescent albumin. MEASUREMENTS AND MAIN RESULTS: Soluble L-selectin significantly attenuated tumor necrosis factor-alpha-mediated increases in leukocyte adherence and vascular leakage. Leukocyte rolling velocity was restored to baseline with soluble L-selectin; however, rolling flux was not altered. Blood pressure, shear rate, and leukocyte counts did not differ between groups. CONCLUSIONS: Soluble L-selectin decreases local inflammation-mediated leukocyte adherence and vascular leakage in vivo. The increased concentrations of soluble L-selectin in sepsis may represent a protective mechanism by which the host attempts to diminish the deleterious systemic effects of activated leukocytes during sepsis.

Dramatic increase in lymph node dendritic cell number during infection by the mouse mammary tumor virus occurs by a CD62L-dependent blood-borne DC recruitment.

Despite the information dealing with the differential phenotype and function of the main mouse dendritic cell (DC) subpopulations, namely, CD8alpha(-) and CD8alpha(+) DCs, their origin and involvement in antiviral immune responses in vivo are still largely unknown. To address these issues, this study used the changes occurring in DC subpopulations during the experimental infection by the Swiss (SW) strain of the mouse mammary tumor virus (MMTV). MMTV(SW) induced an 18-fold increase in lymph node DCs, which can be blocked by anti-CD62L treatment, concomitant with the presence of high numbers of DCs in the outer cortex, in close association with high endothelial venules. These data suggest that the DC increase caused by MMTV(SW) infection results from the recruitment of blood-borne DCs via high endothelial venules, by a CD62L-dependent mechanism. In addition, skin sensitization assays indicate that MMTV(SW) infection inhibits epidermal Langerhans cell migration to the draining lymph node. Moreover, data on the kinetics of MMTV(SW)-induced expansion of the different DC subsets support the hypothesis that CD8(-) and CD8(+) DCs represent different maturation stages of the same DC population, rather than myeloid- and lymphoid-derived DCs, respectively, as previously proposed. Finally, the fact that DCs were infected by MMTV(SW) suggests their participation in the early phases of infection.

Important roles for L-selectin and ICAM-1 in the development of allergic airway inflammation in asthma.

Airway inflammation and airway hyperresponsiveness (AHR) are fundamental features of asthma. Migration of inflammatory cells from the circulation into the lungs is dependent upon adhesion molecule interactions. The cell surface adhesion molecules L-selectin and intercellular adhesion molecule (ICAM)-1 have been demonstrated to mediate leukocyte rolling on inflamed pulmonary endothelium, and ICAM-1 has also been shown to mediate capillary sequestration in inflamed lung. However, their roles in the development of airway inflammation and AHR in asthma have not been directly examined. We have characterised the roles of L-selectin and ICAM-1 in the recruitment of inflammatory cells to the lung and in the development of airway hyperresponsiveness using an ovalbumin (OVA)-induced allergic airway disease model of asthma and adhesion molecule-deficient mice. OVA-sensitized/challenged ICAM-1-deficient mice have dramatically reduced inflammatory influx into the airway/lung and a corresponding attenuation of AHR as compared to wild-type controls. OVA-sensitized/challenged L-selectin-deficient mice demonstrate significantly reduced numbers of CD3(+)lymphocytes and increased numbers of B220(+)lymphocytes in BAL as compared to wild-type mice (P< 0.05). However, other parameters of airway/lung inflammation in OVA-sensitized/challenged L-selectin-deficient mice were equivalent to wild-type control mice. Remarkably, despite a fulminant inflammatory response in the airway/lung, AHR was completely abrogated in OVA-sensitized/challenged L-selectin-deficient mice. These findings suggest a crucial role for ICAM-1 in the development of airway inflammation and AHR in asthma. In contrast, L-selectin plays a more selective role in the development of airway hyperresponsiveness but not allergic inflammation in this animal model of asthma. Thus, L-selectin and ICAM-1 represent potential targets for novel asthma therapies specifically aimed at controlling airway inflammation and/or airway hyperresponsiveness.

Dermal and pulmonary inflammatory disease in E-selectin and P-selectin double-null mice is reduced in triple-selectin-null mice.

In the initial phase of an inflammatory response, leukocytes marginate and roll along the endothelial surface as a result of adhesive interactions between molecules on the endothelial cells and leukocytes. To evaluate the role of the 3 selectins (E, L, and P) in leukocyte rolling and emigration, a null mutation for L-selectin was introduced into previously described embryonic stem cells with null mutations in the genes for both E-selectin and P-selectin (E/P double mutants) to produce triple-selectin-null mice (E-selectin, L-selectin, and P-selectin [E/L/P] triple mutants). Triple-selectin homozygous mutant mice are viable and fertile and only rarely develop the severe mucocutaneous infections or pulmonary inflammation characteristic of E/P double-mutant mice. Surface expression of L-selectin was undetectable in triple-mutant mice on fluorescence-activated cell-sorter analysis of peripheral neutrophils. Pathological studies revealed moderate cervical lymphadenopathy and lymphoplasmacytic infiltrate, but these were less extensive than in E/P double-mutant mice. Neutrophil emigration during thioglycolate-induced peritonitis was significantly reduced at 4, 8, and 24 hours (35%, 65%, and 46% of wild-type values, respectively). Intravital microscopy of the cremaster muscle revealed almost no rolling at times up to 6 hours after exteriorization, with or without addition of tumor necrosis factor alpha. The small amount of residual rolling was dependent on alpha(4)-integrin. The occurrence of skin and pulmonary disease in E/P double-mutant mice but not E/L/P triple-mutant mice suggests that deficiency of L-selectin alters the inflammatory response in E/P mutants. (Blood. 2001;98:727-735)

[The adherence of leukocytes mediated by L-selectin to endothelia activated by burn sera].

OBJECTIVE: To study the mediating effects of L-selectin on the adherence of rat PMNs to the endothelia activated by burn sera. METHODS: Pulmonary microvascular endothelial cells (PMEC) of rat were cultured by tissue block method. Rat PMNs were harvested by continuous perfusion. The samples were collected for the detection of the adhering rate of PMEC to the PMNs after being cultured with L-selectin monoclonal antibody (mAb), L-selectin ligand and sulfatide, respectively. RESULTS: The adhering rate of PMNs to PMEC was obviously lowered by the adding of L-selectin mAb and sulfatide. CONCLUSION: The adherence of PMNs to activated PMEC could be mediated by L-selectin.

Effects of an L-selectin antibody on the pulmonary and systemic manifestations of severe smoke inhalation injuries in sheep.

Sheep were treated with either lymphocyte adhesion molecule (LAM)1-3, an antibody against L-selectin, (40 mg 1 hour before smoke inhalation and 35 mg 24 hours after smoke inhalation; n = 6) or equivalent volumes of 0.9% saline solution (n = 6). After the smoke inhalation injuries, the PaO2/FIO2 ratio declined in both groups until 40 hours after the injuries, when a trend toward improvement was noted in the group that received LAM1-3. Lung lymph flow increased in both groups until 36 hours after the smoke inhalation injuries and then significantly decreased in the group that received LAM1-3. Forty-eight hours after the smoke inhalation injuries, there was a significant decrease in the ratio of wet-dry lung weight and in preservation of the reflection coefficient in the group that received LAM1-3 (P < .05). Histopathologic examination showed no differences between the groups in the pulmonary morphology associated with smoke inhalation. A reduction in splanchnic blood flow was noted in the control group (P < .05); this reduction was attenuated by treatment with LAM1-3. The delayed pulmonary effects and improved splanchnic blood flow suggested that LAM1-3 attenuated the development of a systemically induced secondary lung injury rather than of the primary lung injury associated with smoke inhalation.

Low-dose radiotherapy selectively reduces adhesion of peripheral blood mononuclear cells to endothelium in vitro.

BACKGROUND AND PURPOSE: The anti-inflammatory effect of low-dose radiotherapy (LD-RT) still is not understood. The adhesion of leukocytes to endothelial cells (EC) of the vessel wall is the initial event of tissue invasion, and thus, crucially contributes to the regulation of inflammation. We investigated the influence of LD-RT on the adhesion process in vitro. MATERIALS AND METHODS: Isolated peripheral-blood-mononuclear-cells (PBMC) were incubated with an activated murine endothelioma cell-line under shear conditions at 4 degrees C after irradiation with single doses between 0.1 and 10.0 Gy. Adherent cells were counted microscopically and compared to a non-irradiated control. In parallel, viability and expression of adhesion molecules, especially of L-selectin, and lineage-specific markers on the cell surface were determined by dye exclusion and cytofluorometry, respectively. Modulation of adhesion by soluble L-selectin was tested in the adhesion assay. RESULTS: Radiation doses of 0.1-0.5 Gy reduced the adhesion of viable PBMC to EC in vitro by 70% of the control level 4 h after irradiation. Leukocytes showed a marked reduction of L-selectin expression after LD-RT. Soluble L-selectin can inhibit the adhesion of PBMC to EC. CONCLUSION: The anti-inflammatory effect of LD-RT might, in part, be due to the reduction in the adhesion of PBMC to EC. This reduction in adhesion might be a consequence of the reduced expression of L-selectin on the surface of PBMC, and the inhibition of adherence by soluble L-selectin shed by PBMC in vitro.

Role of selectins on IgE-mediated skin reaction.

Selectins play an important role on leukocytes infiltration into inflammatory tissues. To understand the role of selectins, we investigated the effects of selectin-IgG chimeras and anti selectin antibodies on the murine IgE-mediated skin inflammation model. Biphasic skin reactions were induced by intradermal challenge with ovalbumin (OA) to ears of actively sensitized mice. This reaction was characterized by immediate and late phase responses observed as which were induced via a rapid increase in capillary permeability and leukocyte infiltration, respectively. The expression of E-selectin mRNA was significantly increased to reach its highest level at 2 h after OA challenge. E-, P-, and L-selectin-IgG chimeras inhibited the late phase responses, i.e. ear swelling, neutrophil infiltration and eosinophil infiltration at 24 h after OA challenge in a dose-dependent manner at dose range of 0.1 - 10 mg kg(-1), i.v. Antiselectin antibodies did not inhibit the increase of ear swelling. But anti E- and P-selectin antibodies significantly inhibited neutrophil infiltration and eosinophil infiltration. These results indicate that selectins play an important role on the late phase response of the murine IgE-mediated skin inflammation model by mediating inflammatory cell adhesion to endothelium.

Investigation of the mechanism of lymphocyte injection therapy in treatment of lymphedema with special emphasis on cell adhesion molecule (L-selectin).

We previously employed intraarterial lymphocyte injection therapy in conjunction with standard non-operative treatment of peripheral lymphedema of various etiologies. In this study, we further evaluated the clinical outcome of this therapy in 46 patients with unilateral lymphedema of the extremities. The results showed combined therapy (lymphocyte injection with compression) was effective in 74% (34 of 46 patients) with dramatic reduction in lymphedema in 37% (17 of 46 patients). In the most recent 5 patients treated, we examined the expression of cell adhesion molecule of the lymphocytes (L-selectin) before, during and after lymphocyte injection therapy to study the putative pathomechanism of this treatment method. The expression of L-selectin, a lymphocyte-specific adhesion molecule, increased in the autologous lymphocytes obtained by a blood cell separator and in the lymphocytes from the peripheral blood after injection. Moreover, the lymphocyte fraction, which was positive for L-selectin and negative for CD3, a T-cell marker, decreased after lymphocyte injection. We postulate that the lymphocytes of L-selectin (+) and CD (-) remain in the affected swollen limb and play a role in an ill-defined immunologic responsiveness that potentiates reduction in edema.

L-selectin stimulates the neutral sphingomyelinase and induces release of ceramide.

Selectins have been shown to be crucial in the rolling process of leukocytes during lymphocyte homing and in the early phase of inflammatory processes. Recently, we and others have shown that binding of L-selectin to its ligands correlates with a rapid induction of several intracellular signaling molecules, in particular, Src-like tyrosine kinases, MAP-kinases, Jun NH2-terminal kinase, the small G-proteins Ras and Rac, and a release of Ca2+ in leukocytes. Here, we demonstrate the activation of a novel signaling pathway by L-selectin. Stimulation of Jurkat T-lymphocytes via L-selectin results in an increase of neutral sphingomyelinase activity. This activity correlates with a consumption of cellular sphingomyelin and a release of ceramide. The activation of the neutral sphingomyelinase by L-selectin does not depend on tyrosine kinase activity and, therefore, represents an alternative and novel pathway to stimulate lymphocytes via L-selectin.

Role of primary and secondary capture for leukocyte accumulation in vivo.

Leukocyte accumulation during inflammation depends on the concerted action of selectin and integrin adhesion molecules, which promote capture, rolling, and arrest of these cells on activated endothelium. In addition to interacting with endothelial cells, leukocytes can also adhere to already adherent leukocytes through an L-selectin-dependent mechanism. Initiation of adhesion through this mechanism has been called nucleation and leads to characteristic geometric patterns (ie, clusters and strings) of adherent leukocytes in flow chambers. We have used intravital microscopy of tumor necrosis factor-alpha (TNF-alpha)-treated mouse cremaster muscles to quantitatively investigate the potential role of leukocyte-leukocyte adhesion in initiating and maintaining the leukocyte clusters that are commonly observed in inflamed venules. Our data show that in TNF-alpha-treated venules with diameters between 23 and 108 microm, leukocyte adhesion occurs in clusters that are 19 to 50 microm long and 8 to 44 microm wide. They are almost entirely made up of slow-rolling leukocytes. Of all leukocytes recruited into a cluster (100%), the majority enter the cluster rolling along the endothelium and sharply reduce their velocity in the absence (59%) or presence (15%) of other leukocytes in proximity (one cell diameter). Some of the rolling leukocytes (17%) pass through the cluster without reducing their velocity. Recruitment of leukocytes from the free flow regime into a cluster is a rare event and accounts for only 7 (1.2%) of 476 leukocytes arriving in the cluster. However, of the leukocytes captured from the free flow, 6 initiated contact with a slow-rolling leukocyte rather than making direct contact with the endothelium. Our data show that leukocyte-leukocyte interactions can occur in vivo but are not important for cluster formation. This is confirmed by the observation of normal cluster formation in L-selectin-deficient mice, in which leukocyte-leukocyte interactions under flow are abolished. We conclude that leukocyte-mediated nucleation contributes little to leukocyte recruitment during inflammation in vivo. Cluster formation appears to be dominated by areas of endothelium with a higher expression of E-selectin, because cluster formation is greatly reduced in E-selectin-deficient mice.

Synthesis of a new nanomolar saccharide inhibitor of lymphocyte adhesion: different polylactosamine backbones present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode.

Lymphocyte infiltration is a hallmark of acute rejections in solid organ transplants, such as cardiac allograft. We have previously shown that lymphocyte extravasation to cardiac grafts undergoing rejection is largely due to interactions between lymphocyte L-selectin and its sialyl Lewis x (sLex) decorated ligands. Our previous work demonstrated further that an enzymatically synthetized tetravalent sLex glycan of a branched polylactosamine backbone is a highly efficient inhibitor of L-selectin-dependent lymphocyte adhesion to graft endothelium. To improve the availability of multivalent sLex glycans for anti-inflammatory indications, we now report enzymatic synthesis of another tetravalent sLex glycan that can be potentially produced on a large scale, and show that even the new saccharide is a nanomolar inhibitor of L-selectin-dependent lymphocyte adhesion. The novel antagonist is sLex beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc (8) (where LacNAc is the disaccharide Gal beta 1-4GlcNac and sLex is the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc). Its five-step synthesis was started from the octameric polylactosamine LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc (3), which in turn is accessible in one step from the hexasaccharide LacNAc beta 1-3'LacNAc beta 1-3'LacNAc. Importantly, the hexasaccharide primer has been synthesized chemically (Alais and Veyrieres, Tetrahedron Lett., 24, 5223, 1983). Hence, our data outline a route to glycan 8, consisting of a combination of chemical and enzymatic methods of oligosaccharide synthesis. In addition, our data show that polylactosamine backbones are able to present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode, without a requirement for uniqueness in the backbone structure.

Tumor necrosis factor-alpha induces leukocyte recruitment by different mechanisms in vivo and in vitro.

It is well established that E-selectin is the endothelial adhesion molecule that is primarily responsible for mediating leukocyte rolling on TNF-alpha-stimulated cultured endothelial cells. Despite this, few studies in in vivo inflammatory models have observed reduced leukocyte accumulation using mAbs against E-selectin. The objective of this study was to compare the function of E-selectin on endothelial cells in vitro with its role in TNF-alpha-induced leukocyte recruitment in vivo using EL246, a mAb that blocks the function of E-selectin on activated feline endothelial cells. In vitro experiments using feline endothelial cells showed that EL246 functionally inhibits E-selectin-dependent leukocyte recruitment induced by TNF-alpha, without affecting the function of other rolling mechanisms. Intravital microscopy of single 25- to 40-microm venules in the feline mesentery was then used to examine leukocyte rolling and adhesion in response to superfusion with TNF-alpha. TNF-alpha treatment significantly increased the number of both rolling and adherent leukocytes and significantly decreased leukocyte rolling velocity. Treatment with EL246 (1 mg/kg), either i.v. at the start of the TNF-alpha protocol or directly into the superior mesenteric artery after 3 h of TNF-alpha treatment, had no effect on leukocyte rolling, adhesion, or rolling velocity. However, treatment with the selectin-binding carbohydrate, fucoidan, reduced leukocyte rolling to below baseline levels. These results suggest that in contrast to its prominent role on cultured endothelial cells, E-selectin does not contribute to leukocyte recruitment in TNF-alpha-stimulated feline mesenteric venules in vivo.

Neutrophil CD18-dependent arrest on intercellular adhesion molecule 1 (ICAM-1) in shear flow can be activated through L-selectin.

Neutrophil emigration through endothelial cells under shear flow involves several adhesion processes including cell rolling, arrest, and transmigration. Rolling is mediated by selectins, while arrest and transmigration both require activated CD18 integrins. One mode of CD18 activation is via selectins expressed on neutrophils and endothelial cells. We have recently reported that cross-linking of L-selectin (CD62L) resulted in the rapid activation of CD18-dependent adhesion. In the current study, we examine whether binding of E-selectin (CD62E) and L-selectin can activate neutrophil CD18-dependent adhesion under shear flow. Human ICAM-1 (CD54) and E-selectin were co-transfected into L cells. Neutrophil capture, rolling, and arrest on these monolayers were quantitated in a parallel plate flow chamber at a wall shear stress of 2.0 dyne/cm2. Under these conditions, E-selectin supported cell capture and rolling on the monolayer, but did not trigger CD18-mediated cell arrest within 200 microm of rolling. However, when neutrophils were treated with anti-L-selectin mAb and cross-linked with a secondary mAb, approximately 50% of the cells arrested within 54 microm. Cell arrest was also observed in response to IL-8 stimulation. A subthreshold level of IL-8 in combination with L-selectin cross-linking potentiated the level of cell arrest due to either stimulus alone. The transition to cell arrest involved both LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). Blocking either subunit alone failed to reduce arrest, while blocking both molecules with mAbs reduced the number to baseline levels. These data support the conclusion that L-selectin, but not E-selectin, can signal the transition from neutrophil rolling to cell arrest under shear flow.

L-selectin activates JNK via src-like tyrosine kinases and the small G-protein Rac.

Selectin and alpha 4 beta 7-integrins have been shown to mediate transient leucocyte interactions with endothelial cells which is a crucial step in the initial immune response to pathogens. We have previously shown that stimulation of T lymphocytes via L-selectin results in activation of a signalling cascade from the L-selectin molecule via the tyrosine kinase p56lck and tyrosine phosphorylation of L-selectin to the stimulation of p21Ras and Rac proteins. In the present study we demonstrate that stimulation of Jurkat T lymphocytes via L-selectin results in an activation of Jun N-terminal kinase (JNK) but not of p38-K. L-selectin-initiated activation of JNK is mediated by src-like tyrosine kinases and the small G-protein Rac 1/2, since genetic or pharmacological inhibition of p56lck or Rac proteins prevent the stimulation of JNK by L-selectin. Thus, the data point to a novel signalling cascade from L-selectin via src-like tyrosine kinases and Rac proteins to JNK.

Cell adhesion molecules of experimental otitis media in the rat.

We examined the expression of cell adhesion molecules (CAM) in immune-mediated otitis media using keyhole limpet haemocyanin (KLH) in the rat, as well as the regulation of these CAM in peripheral blood polymorphonuclear leucocytes (PMN) and lymphocytes upon exposure to middle ear effusion (MEE). After general immunization, a topical antigen was introduced into the middle ear cavity. One day after exposure, CD18+ cells, primarily PMN, had maximally invaded the middle ear mucosa and mucosal epithelium. Mucosal epithelium strongly expressed intercellular adhesion molecule-1 (ICAM-1), only on the first day. The total number of cells in MEE reached a peak on day 3. On day 3, ICAM-1+ cells had reached a peak of 24.5% of the total cells. On day 2. CD18+ cells had reached a peak at 75.3% of the total cells. We examined the regulation of CAM in peripheral blood upon exposure to MEE. The percentage of fluorescent CD18+ PMN increased with MEE compared to those incubated in its absence, but those of L-selectin-positive PMN significantly decreased CAM on the surface lymphocytes did not change when incubated with MEE. The expression of CAM (CD18, ICAM-1) appears important for the initiation of otitis media. Moreover, it was thought that the interaction between the infiltrated PMN and MEE may modify the expression of CAM during the inflammatory process in the middle ear cavity.