Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have investigated the role of tumor necrosis factor (TNF) in contributing to eosinophil recruitment in a mouse model of asthma. TNF is one of several proinflammatory cytokines released into the airway during episodes of asthma (1). Increased levels of TNF messenger RNA (mRNA) and protein have been measured in bronchoalveolar lavage (BAL) cells and fluid derived from asthma patients with symptoms compared with asymptomatic asthmatics (1). Inhalation of TNF induces airway inflammation and bronchial hyperresponsiveness in rats (4), sheep (5), and normal human subjects (6). The potential cellular source(s) of TNF in asthma includes macrophages (7), mast cells (2, 7, 8), and eosinophils (9). The ability of allergen-activated immunoglobulin (Ig) E receptors to mediate TNF expression is suggested from studies in which anti-IgE treatment of human lung tissue resulted in the upregulation of TNF mRNA expression (7, 10). In addition, IgE-dependent stimulation of alveolar macrophages derived from allergic asthmatics generates greater amounts of TNF compared with alveolar macrophages derived from control subjects (11). These studies all suggest a potential important role for TNF in the pathogenesis of asthma.

TNF is first produced as a cell membrane-bound 26-kD molecule that is cleaved from the cell surface by a TNF- converting enzyme to generate a secreted 17-kD form of the TNF molecule (12). Membrane as well as secreted forms of TNF trimerize and interact as homotrimers with two structurally related TNF receptors (TNF-Rs), namely TNF-RI (mouse p55, human p60) and TNF-RII (mouse p75; human p80), which are functionally distinct (12). Both the TNF-RI and the TNF-RII are coexpressed on the surface of most cell types (12, 13). The TNF-RI mediates the majority of proinflammatory responses attributed to TNF (12, 13), whereas the TNF-RII mediates TNF-induced thymocyte proliferation (14) and TNF-induced apoptosis of activated T lymphoctes (12, 14). Both TNF-R are initially integral membrane proteins but can be cleaved from the cell surface to become bioactive soluble receptors (12).

TNF released at sites of allergic inflammation may contribute to leukocyte recruitment from the circulation by several mechanisms, including induction of endothelial cell adhesion molecule expression. TNF is an important stimulus for the expression of endothelial cell adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, E-selectin, and, in the mouse, P-selectin (15). Intravital microscopy studies of the TNF-treated feline mesenteric circulation have demonstrated significant increases in the number of circulating leukocytes rolling and adherent to TNF-treated vascular endothelium (18). This effect of TNF on endothelial cell adhesion molecule expression is mediated through the TNF-RI as evidenced by in vivo studies demonstrating reduced expression of these adhesion molecules, including VCAM-1 and E-selectin in TNF-challenged TNF-RI-deficient mice (16). Furthermore, the biologic significance of the reduced level of adhesion molecule expression in TNF-RI- deficient mice is underscored by the fact that TNF-triggered mononuclear cell and neutrophil infiltration of lung, liver, and kidney is inhibited in TNF-RI-deficient mice compared with wild-type mice (16). Studies using TNF-RI- and TNF-RII-deficient fibroblasts demonstrate that TNF-induced adhesion molecule expression (ICAM-1 and VCAM-1) is mediated by only the TNF-R1 and not the TNF-RII (19).

As we were interested in determining the relative importance of TNF compared with other cytokines in mediating eosinophil adhesion to endothelium and eosinophil tissue recruitment in vivo, we used TNF-RI/TNF-RII double knockout mice (i.e., TNF-R p55/p75 [TNF-R55/75]- deficient mice) as well as TNF-RI knockout mice (i.e., TNF-R p55 [TNF-R55]-deficient mice) to investigate the role of TNF in the recruitment of eosinophils to tissue sites challenged with allergen.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

TNF-R55/75- and TNF-R55-Deficient Mice

TNF-R55/75- and TNF-R55-deficient mice were kindly provided by Dr. J. Peschon (Immunex, Seattle, WA) (20). Both TNF-R55/ 75- and TNF-R55-deficient mice are overtly normal and exhibit normal development and homeostasis under basal conditions (20). Thymopoesis, lymph node development, and the total lymphoid cellularity of the thymus, spleen and mesenteric lymph nodes are similar in TNF-R55/75-deficient and control mice (20). Selective deficits in several TNF-mediated inflammatory responses (Listeria monocytogenes infection and endotoxic shock) have been noted in both TNF-R55/75- and TNF-R55-deficient mice (20). C57Bl/6 × 129J hybrid background wild-type female mice, 8 to 10 wk of age, were used as controls.

Mouse Model of Eosinophilic Pulmonary Inflammation

TNF-R55/75-deficient mice. Pulmonary eosinophilia in mice was induced as previously described in this laboratory (21). In brief, wild-type or TNF-R55/75-deficient mice were sensitized by intraperitoneal injection of 50 µg ovalbumin (OVA)/1 mg alhydrogel (Aldrich Chemical Co., Milwaukee, WI) in 0.9% sterile saline on Days 0 and 12. Nonsensitized mice received 1 mg of alhydrogel in 0.9% saline. On Day 24, the appropriate groups of mice (n = 4 mice/group) were exposed three times (at 1-h intervals) to an aerosol of OVA (10 mg/ml) in 0.9% saline (nonsensitized mice received saline only) for 30 min. The aerosol is generated at 6 liters/min by a nebulizer (Ultra-Neb 99; Devilbiss, Somerset, PA) into a closed chamber of 800 cm³. The aerosolized OVA protocol was then repeated on Days 26, 28, and 30. Three hours after the last aeroallergen challenge, mice were killed by CO₂ asphyxiation.

TNF-R55-deficient mice. TNF-R55-deficient mice and wild-type control mice were sensitized to OVA as described previously for experiments with TNF-R55/75-deficient mice. Studies with TNF-R55-deficient mice were performed as indicated previosuly for TNF-R55/75-deficient mice other than the OVA airway challenges performed on Days 24, 26, 28, and 30, which were performed intranasally (10 µg OVA in 50 µl phosphate-buffered saline [PBS]) as opposed to using a nebulizer. The intranasal route of OVA administration induces a stronger BAL eosinophilic response in wild-type mice (~ 85% BAL eosinophils) compared with the inhalation route with a nebulizer (~ 37% BAL eosinophils) and allowed us to also evaluate the contribution of TNF after a stronger airway antigen challenge than that induced by aerosolized antigen.

BAL Cells

BAL cells from wild-type, TNF-R55/75-deficient, and TNF-R55- deficient mice were recovered by lavage with 1 ml of PBS via a tracheal catheter. The resulting BAL cells were immediately separated from BAL fluid by centrifugation (700 × g for 5 min). An appropriate PBS dilution of the recovered BAL cells was added to trypan blue, and the viability and total number of BAL white blood cells were counted with a hemocytometer. Differential leukocyte counts were performed after brief acetone fixation and staining of the BAL cells with May-Grünwald-Giemsa stains. The percentage of eosinophils, neutrophils, and mononuclear cells present on each slide was assessed by counting a minimum of 300 cells in random high power fields using a light microscope (magnification: ×40).

Lung Tissue Eosinophils

Lung tissue from wild-type and TNF-R55-deficient mice (embedded in OCT in 10 × 50 × 50-mm tissue wells) was cryosectioned at 10 µm and acetone-fixed onto poly (L-lysine)-coated slides. Total eosinophil numbers were enumerated by detection of eosinophil peroxidase using diaminobenzidine tetrahydrochloride (DAB) staining and microscopic examination, as described in this laboratory (23). Slides were incubated at room temperature for 1 min in the presence of cyanide buffer (10 mM potassium cyanide, pH 6), rinsed in PBS, and incubated for 10 min with the peroxidase substrate DAB (Vector Lab, Burlingame, CA). Slides were subsequently washed in PBS, counterstained with hematoxylin, air-dried, and examined by light microscopy (magnification ×40). Five random fields were selected and eosinophils were counted (cells staining brown) to determine total eosinophil number per microscope field.

Peripheral Blood Eosinophils

Blood was collected from the carotid artery. Red blood cells were lysed using a 1:10 solution of 100 mM potassium carbonate, 1.5 M ammonium chloride. The remaining cells were cytospun (3 min at 500 rpm) onto microscope slides and air-dried. Eosinophil counts were performed as described previously.

Determination of Airway Responsiveness to Methacholine

Airway responsiveness was assessed on Day 30, after the final OVA inhalation, using a single chamber whole body plethysmograph obtained from Buxco (Troy, NY), as previously described (23). In this system, an unrestrained, spontaneously breathing mouse is placed into the main chamber of the plethysmograph, and pressure differences between this chamber and a reference chamber are recorded. In the plethysmograph, mice were exposed for 3 min to nebulized PBS and subsequently to increasing concentrations of nebulized methacholine (MCh) (Sigma, St. Louis, MO) in PBS using an Aerosonic ultrasonic nebulizer (DeVilbiss). After each nebulization, recordings were taken for 3 min. The Penh values were measured during each 3-min sequence and are expressed as the mean for each MCh concentration (23).

Immediate Hypersensitivity Skin Test

Wild-type and TNF-R55/75-deficient mice were sensitized to OVA as described previously. On Day 30, a total of 50 µl of OVA antigen or diluent control was injected into the shaved backs of the different groups of mice. Immediately after antigen administration, a total of 200 µl of 1% Evans blue dye was injected into the tail vein of the mice (22, 24). The size of blueing of the skin (measured as the largest transverse diameter, in millimeters) at the challenged site was assessed 15 min later.

Mouse Model of Peritoneal Eosinophilic Inflammation: Ragweed Allergen Immunization and Peritoneal Allergen Challenge

The techniques used for ragweed immunization and challenge are as previously described in this laboratory (22, 25). Mice were immunized by a series of five injections of a 1:1,000 dilution of a ragweed pollen extract (Miles Inc., Spokane, WA). A total of 0.1 ml was injected subcutaneously on Days 0 and 1, and 0.2 ml was injected subcutaneously on Days 6, 8, and 14. A control group of ragweed-immunized mice (challenged with PBS diluent) and nonimmunized mice (prepared by subcutaneous injections of isotonic saline instead of the ragweed pollen extract) followed the same immunization schedule. Three to five mice were included in each group of mice studied. The mice were challenged on Day 20 by the intraperitoneal injection of 0.2 ml of the ragweed allergen (or control PBS diluent).

Assessment of Eosinophils in the Peritoneal Cavity

At time points before (Day 0) and after immunization, as well as before and 48 h after intraperitoneal allergen challenge (Day 22), the mice were killed by cervical dislocation. A total of 2 ml of PBS containing 6 U/ml of heparin was injected intraperitoneally, the abdomen massaged, and the peritoneal infusion collected after the peritoneum was opened. An appropriate PBS dilution of the recovered peritoneal fluid was added to trypan blue, and the viability and total number of white blood cells were counted with a hemocytometer. Differential leukocyte counts were performed after brief acetone fixation and staining of the peritoneal cells with May-Grünwald-Giemsa stains. The percentage of eosinophils present on each slide was assessed by counting a minimum of 300 cells in random high power fields using a light microscope (magnification: ×40).

Isolation of Murine Eosinophils from Interleukin-5 Transgenic Mice for Fluorescent Labeling

Mouse eosinophils of 85 to 95% purity and > 98% viability were purified from interleukin (IL)-5 transgenic mice (kindly provided by Dr. Colin Sanderson, TVW Telethon Institute for Child Health Research, West Perth, Australia) (26) using a percoll gradient as previously described (22, 25). IL-5 transgenic mice (10 wk of age) had peripheral blood leukocyte differential cell counts exhibiting ~ 40 to 60% eosinophils. The remaining white blood cells comprised ~ 30 to 40% T lymphocytes, ~ 2% mononuclear cells, and ~ 10% neutrophils. Eosinophils with at least 98% viability and > 90% purity were selected and labeled with carboxy fluorescein diacetate (CFDA) (Molecular Probes, Eugene, OR) as previously described in our laboratory for labeling of murine and human eosinophils (22, 25, 27). CFDA-labeled eosinophils were resuspended at a concentration of 0.5 × 10⁷ cells/200 µl of PBS containing 0.01% glucose and kept at room temperature in the dark until used.

Fluorescence-Activated Cell Sorter Analysis of Mouse Eosinophil Adhesion Molecule Expression

Mouse eosinophils derived from IL-5 transgenic mice were incubated with the primary monoclonal antibodies (mAbs) MEL-14 (a rat antimouse L-selectin IgG_2a mAb), PS/2 (a rat antimouse 4 integrin IgG_2b mAb), or species- and isotype-matched mAbs on ice for 30 min, washed, and incubated with goat antirat fluorescein isothiocyanate IgG (Sigma) for an additional 30 min. Surface expression was determined on a fluorescence-activated cell sorter (FACS)star flow cytometer (Becton Dickinson, Mountain View, CA) (27) after gating for eosinophils by characteristic foreward and side light scatter.

Preparation of Mice for Detection of Eosinophil Rolling and Adhesion in the Peritoneal Microcirculation

TNF-R55/75-deficient, TNF-R55-deficient, or control wild-type mice (25 to 35 g body weight) that had been ragweed sensitized were prepared for intravital microscopy 24 h after the final peritoneal ragweed or PBS challenge as previously described (22, 25). We have previously reported (22, 25) that this 24-h post-allergen challenge time point is an optimal time point for visualizing allergen-induced intravascular eosinophil rolling and adhesion in this mouse model. The 24-h time point was chosen as it slightly precedes peak eosinophil tissue recruitment, which peaks 24 to 48 h after allergen challenge in this model (22, 25, 28). Mice were anesthetized with a subcutaneous injection of saline solution containing a cocktail of ketamine hydrochloride and xylazine (7.5 mg and 2.5 mg, respectively, per 100 mg body weight). The mice were then placed on a heating pad maintained at 37°C. A midline incision was made and the mesentery was gently exteriorized and spread on a heated glass window (37.5°C) of the stage of a Leitz (Wetzler, Germany) intravital microscope. The exteriorized portion of mouse mesentery was kept continuously moist with endotoxin-free isotonic saline solution (pH 7.4). Other parts of the intestine that were exposed but not microscopically observed were kept moist with isotonic saline-soaked cotton pads and the mesentery was covered with Saran Wrap. To minimize endotoxin contamination, Saran Wrap was presoaked with 1% E-Toxa-Clean (Sigma) overnight, followed by rinsing in 70% ethanol, endotoxin-free distilled water, and a final wash with sterile isotonic saline solution.

Visualization of Eosinophils in the Mouse Mesentery

Fluorescent CFDA-labeled eosinophils were injected into the tail vein of mice previously sensitized with ragweed allergen and challenged with either ragweed or saline 24 h before intravital fluorescence microscopy as previously described (22, 25). All studies were conducted between 0 and 2 h after exteriorization of the mouse mesentery. The rolling of mouse eosinophils in mesenteric venules was made visible by stroboscopic epi-illumination using a video-triggered Xenon lamp and Leitz Ploemopak epi-illuminator employing an I2 filter block. All images were recorded through a silicon-intensified tube camera (SIT68; Dage MTI, Michigan City, IN) using a 10× or 20× water immersion objective (Nikon, Melville, NY) as described previously (22, 25). The rolling fraction of CFDA-labeled mouse eosinophils in ragweed-challenged mice (wild-type control, TNF-R55/75-deficient, and TNF-R55-deficient mice) was determined by frame-by-frame analysis as previously described (22, 25). Rolling eosinophils were quantitated by counting the number of eosinophils interacting with the vessel wall in 1 min in a plane perpendicular to a vessel axis, whereas those cells that were found to be stationary for at least 1 min were considered as adherent eosinophils.

Statistics

The number of eosinophils in BAL fluid and peritoneal cavity were compared by Student's t test using a statistical software package (In Stat, San Diego, CA). Rolling fractions of injected eosinophils were compared by multiple comparisons of paired data by Student's t test using a statistical software package (SigmaStat; Jandel Scientific, San Rafael, CA). P values of < 0.05 were considered statistically significant. All results are given as mean ± standard error of the mean.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Mouse Model of Eosinophilic Lung Inflammation

TNF-R55/75-deficient mice. Sensitization and OVA allergen challenge of wild-type mice (n = 3 experiments) induced a significant BAL eosinophilia (37.1 ± 6.7% BAL eosinophils) compared with mice that were not sensitized or challenged with OVA (1.8 ± 0.5% BAL eosinophils; P = 0.003) or compared with mice immunized with OVA and challenged with PBS diluent (5.7 ± 1.4% BAL eosinophils; P = 0.002 (Figure 1A). Neutrophils composed less than 2% of BAL cells preallergen, postallergen, or postdiluent challenge. Mononuclear cells composed the remainder of the BAL cells.

View larger version (12K): [in this window] [in a new window]   View larger version (16K): [in this window] [in a new window]   Figure 1.   Quantitative analysis of BAL eosinophils in TNF-R- deficient mice. (A) TNF-R55/75-deficient mice: The number of eosinophils in BAL fluid was enumerated pre- and postallergen challenge, and postdiluent challenge in wild-type (WT) and TNF-R55/75-deficient mice. Results are expressed as the percentage of eosinophils in BAL fluid (n = 3 experiments). There was a significant inhibition of BAL eosinophilia in TNF-R55/75-deficient mice postallergen challenge versus wild-type mice postallergen challenge (P = 0.03). (B) TNF-R55-deficient mice: The number of eosinophils in BAL fluid was enumerated pre- and postintranasal allergen challenge (as opposed to inhalation allergen challenge in TNF-R55/75-deficient mice) in wild-type (WT) and TNF-R55-deficient mice. Results are expressed as the percentage of eosinophils in BAL fluid (n = 3 experiments). There was a significant inhibition of BAL eosinophilia in TNF-R55-deficient mice postallergen challenge versus wild-type mice postallergen challenge (P = 0.05).

In contrast to wild-type mice, TNF-R55/75-deficient mice (n =3 experiments) immunized and challenged with OVA developed significantly less BAL eosinophilia (TNF-R55/75- deficient mice, 12.5 ± 2.5% BAL eosinophils versus wild-type mice, 37.1 ± 6.7% BAL eosinophils; P = 0.03) (Figure 1A). Neither TNF-R55/75-deficient mice that were not sensitized and challenged with OVA (0 ± 0% BAL eosinophils) nor TNF-R55/75-deficient mice immunized with OVA and challenged with PBS diluent (1.0 ± 0.6% BAL eosinophils) developed significant BAL eosinophilia.

To determine whether differences in sensitization to antigen could account for the differences in eosinophil recruitment between wild-type and TNF-R55/75-deficient mice, we performed immediate hypersensitivity skin testing in the different groups of mice. Wild-type and TNF-R55/75-deficient mice sensitized with OVA and challenged intradermally with OVA developed equivalent-sized immediate hypersensitivity skin blueing reactions when Evans blue dye was injected into the tail vein of these mice (wild-type mice, 7 ± 1 mm; TNF-R55/75-deficient mice, 6 ± 1 mm) (n = 3).

TNF-R55-deficient mice. TNF-R55-deficient mice (n = 3 experiments) immunized and challenged with OVA developed significantly less BAL eosinophilia (TNF-R55- deficient mice, 50.8 ± 11.0% BAL eosinophils versus wild-type mice, 85.0 ± 1.9% BAL eosinophils; P = 0.05 (Figure 1B) compared with wild-type mice. Neither TNF-R55-deficient mice that were not sensitized and challenged with OVA (0 ± 0% BAL eosinophils) nor TNF-R55-deficient mice immunized with OVA and challenged with PBS diluent (0.8 ± 0.4% BAL eosinophils) developed significant BAL eosinophilia.

The number of lung eosinophils was also significantly reduced in TNF-R55-deficient compared with wild-type mice (TNF-R55-deficient mice, 9.9 ± 2.6 lung eosinophils/ hpf versus wild-type mice, 33.0 ± 4.3 lung eosinophils/hpf, n = 3; P = 0.001 (Figure 2). There were few eosinophils pre-allergen challenge in the lungs of either wild-type mice (0.3 ± 0.2 eosinophils/hpf) or TNF-R55-deficient mice (0 ± 0 eosinophils/hpf) (Figure 2).

View larger version (15K): [in this window] [in a new window]   Figure 2.   Quantitative analysis of lung eosinophils in TNF-R-deficient mice. The number of lung eosinophils was enumerated pre- and postintranasal allergen challenge in wild-type (WT) and TNF-R55- deficient mice. Results are expressed as the number of lung eosinophils/hpf (n = 3 experiments). There was a significant inhibition of lung eosinophilia in TNF-R55-deficient mice postallergen challenge versus wild-type mice postallergen challenge (P = 0.001).

The percentage of peripheral blood eosinophils in TNF-R55-deficient mice (5.2 ± 0.7% blood eosinophils) postallergen challenge was slightly, but not statistically significantly, greater than that in wild-type mice postallergen challenge (3.2 ± 0.9% blood eosinophils, n = 3; P = not significant).

Airway Responsiveness to MCh

There was no significant difference in airway hyperresponsiveness to MCh in TNF-R55-deficient mice compared with wild-type mice (n = 3; P = not significant) (Figure 3). It should be noted that although eosinophil recruitment was inhibited by 41% in TNF-R55-deficient mice, these mice still had significant levels of BAL eosinophils (51%) and lung eosinophils (10 lung eosinophils/hpf), which could have maintained their airway hyperreactivity to MCh.

View larger version (20K): [in this window] [in a new window]   Figure 3.   Airway hyperreactivity to MCh. Airway hyperreactivity to nebulized PBS or MCh was measured noninvasively on Day 30 after the final OVA challenge in wild-type and TNF-R55-deficient mice using a Buxco plethysmograph (n = 3). Penh values in allergen-challenged TNF-R55-deficient mice were not significantly different from that noted in allergen-challenged wild-type mice at all the concentrations of MCh (3 to 24 mg/ml) tested.

Mouse Model of Eosinophilic Peritonitis

Wild-type mice (n = 3 separate experiments) when immunized and challenged with ragweed allergen developed significantly higher levels of peritoneal cavity eosinophilia (7.1 ± 1.4% eosinophils) compared with wild-type mice that were not challenged with ragweed (0.8 ± 0.6% eosinophils; P = 0.004) or compared with wild-type mice that were immunized with ragweed and challenged with PBS diluent (1.7 ± 0.5% eosinophils; P = 0.005) (Figure 4). In contrast to wild-type mice, TNF-R55/75-deficient mice immunized with ragweed developed significantly less peritoneal eosinophilia when challenged with an intraperitoneal injection of ragweed allergen (TNF-R55/75-deficient mice, 2.9 ± 0.6% peritoneal eosinophils, P = 0.02 versus ragweed-challenged wild-type mice) (Figure 4). There was no significant difference in the number of peritoneal eosinophils in wild-type mice compared with TNF-R55/75-deficient mice that were not immunized or challenged with ragweed (0.8 ± 0.6% peritoneal eosinophils in wild-type versus 0.1 ± 0.1% peritoneal eosinophils in TNF-R55/75-deficient mice).

View larger version (14K): [in this window] [in a new window]   Figure 4.   Eosinophil peritoneal recruitment in TNF-R-deficient and wild-type mice. Ragweed-sensitized mice (TNF-R55/75-deficient or control wild-type mice) (n = 3 experiments) were challenged with an intraperitoneal injection of ragweed allergen. Forty-eight hours later, the percentage of transmigrated peritoneal eosinophils was assessed by light microscopy. TNF-R55/75- deficient mice developed significantly less peritoneal eosinophilia compared with control wild-type mice postallergen challenge (P = 0.004).

Expression of Adhesion Receptors by Mouse Eosinophils

Mouse eosinophils, purified from IL-5 transgenic mice for infusion into the peritoneal microcirculation of allergen- challenged mice, expressed the eosinophil rolling receptors L-selectin and 4 integrin as assessed by FACS analysis (Figure 5).

View larger version (12K): [in this window] [in a new window]   Figure 5.   FACS analysis of mouse eosinophil adhesion molecule expression. Expression of L-selectin (mAb MEL-14, a rat antimouse L-selectin IgG2a mAb)(a and b) and 4 integrins (PS/2, a rat antimouse 4 integrin IgG2b mAb) (c and d) on purified populations of FITC-labeled murine eosinophils derived from IL-5 transgenic mice was determined on a FACSstar flow cytometer.

Intravital Microscopy and Eosinophil Rolling and Adhesion

TNF-R55/75-deficient mice. The passage of the fluorescently labeled eosinophils in the allergen challenged mesenteric circulation was made visible by stroboscopic epi-illumination (Figure 6). We have previously demonstrated that peritoneal ragweed challenge induces a significant increase in eosinophil rolling in the mesenteric venules of wild-type mice challenged with ragweed compared with wild-type mice challenged with PBS diluent (28). The rolling of eosinophils in venules of ragweed-challenged TNF-R55/75- deficient mice (eosinophil rolling fraction, 13.5 ± 7.9%) was found to be significantly reduced compared with ragweed-challenged wild-type mice (eosinophil rolling fraction, 19.7 ± 9.1%; P = 0.02) (Figure 7A).

View larger version (73K): [in this window] [in a new window]   Figure 6.   Eosinophil rolling and firm adhesion visualized by intravital videomicroscopy. Ragweed-sensitized wild-type mice were challenged with an intraperitoneal injection of ragweed. Twenty-four hours after intraperitoneal ragweed allergen challenge, fluorescently labeled eosinophils were injected via the tail vein and visualized in vivo in the peritoneal microcirculation using intravital videomicroscopy. A videotape image of eosinophils flowing through a postcapillary venule is depicted. Blood flow in the venule is from left to right. (B) An image of the same venule depicted in A taken 4 s later. Eosinophils numbered 1 and 3 remain stationary (same positions in A and B), representing eosinophils adherent to the venular endothelium. Eosinophil number 2 transiently rolled on the endothelium and is visible in different positions in A and B.

View larger version (30K): [in this window] [in a new window]   Figure 7.   Comparison of allergen-induced eosinophil rolling and firm adhesion in TNF-R-deficient and control wild-type mice using intravital microscopy. Ragweed-sensitized mice (TNF-R55/ 75-deficient, TNF-R55-deficient, or control wild-type mice) were challenged with an intraperitoneal injection of ragweed. Twenty-four hours later, fluorescently labeled mouse eosinophils were infused intravenously into the mouse tail vein. The mouse mesentery was visualized by intravital microscopy, and intravascular eosinophil rolling and adhesion to endothelium were recorded on a videotape. The number of eosinophils rolling on endothelium in ragweed-challenged TNF-R55/75-deficient, or TNF-R55-deficient mice was significantly less than that observed in ragweed-challenged control wild-type mice (TNF-R55/75 versus wild-type, n = 3; P = 0.02 [A]; and TNF-R55 versus wild-type, n = 3; P = 0.02 [C]). (B) The number of eosinophils adherent to endothelium in ragweed-challenged TNF-R55/75-deficient mice was significantly less than that observed in ragweed-challenged control wild-type mice (TNF-R55/75 versus wild-type, n = 3; P = 0.01) but (D) did not reach statistical significance in TNF-R55-deficient mice (TNF-R55 versus wild-type, n = 3; P = 0.10).

As observed with eosinophil rolling, reduced levels of eosinophil adhesion was observed in TNF-R55/75-deficient mice compared with wild-type mice that were challenged with ragweed (1.1 ± 1.4 adherent eosinophils/250 µm blood vessel length in ragweed challenged TNF-R55/75-deficient mice versus 3.2 ± 0.9 adherent eosinophils/250 µm blood vessel length in ragweed-challenged wild-type mice; P = 0.01) (Figure 7B). The reduced number of firmly adherent eosinophils in TNF-R55/75-deficient mice resulted from an effect both on eosinophil rolling as well as an effect on eosinophil firm adhesion as evidenced by the fact that eosinophil firm adhesion was inhibited as well as eosinophil rolling.

Although allergen challenge induced lower levels of eosinophil rolling and firm adhesion to endothelium in TNF-R55/75-deficient mice compared with wild-type mice, allergen was still able to induce significant eosinophil rolling and firm adhesion in TNF-R55/75-deficient mice as compared to diluent challenge of TNF-R55/75-deficient mice (1.1 ± 1.4 adherent eosinophils after ragweed challenge versus 0.3 ± 0.6 adherent eosinophils postdiluent challenge, P = 0.02; 13.5 ± 7.9 rolling eosinophils after ragweed challenge versus 6.9 ± 6.8 rolling eosinophils postdiluent challenge, P = 0.08) (Figure 8).

View larger version (22K): [in this window] [in a new window]   Figure 8.   Comparison of eosinophil rolling and firm adhesion in TNF-R-deficient mice challenged with allergen versus diluent using intravital microscopy. Ragweed-sensitized mice (TNF-R55/ 75-deficient mice) were challenged with an intraperitoneal injection of ragweed or diluent (n = 3). Twenty-four hours later, fluorescently labeled mouse eosinophils were infused intravenously into the mouse tail vein. The mouse mesentery was visualized by intravital microscopy, and intravascular eosinophil rolling and adhesion to endothelium were recorded on a videotape. (a) The number of eosinophils rolling on endothelium in ragweed-challenged TNF-R55/75-deficient mice was more than that observed in diluent-challenged TNF-R55/75-deficient mice, although this did not quite reach statistical significance (P = 0.08). (b) The number of eosinophils adherent to endothelium in ragweed-challenged TNF-R55/75-deficient mice was significantly more than that observed in diluent-challenged TNF-R55/75-deficient mice (P = 0.02).

TNF-R55-deficient mice. Studies with TNF-R55-deficient mice confirmed that the TNF-R55 was mediating the inhibition of eosinophil rolling and firm adhesion noted in the TNF-R55/75-deficient mice. The rolling of eosinophils in venules of ragweed-challenged TNF-R55-deficient mice (eosinophil rolling fraction, 7.5 ± 2.8%) was found to be significantly reduced compared with ragweed-challenged wild-type mice (eosinophil rolling fraction, 37.3 ± 5.8%, n = 3; P = 0.02) (Figure 7C). As observed with eosinophil rolling, reduced levels of eosinophil adhesion were observed in TNF-R55-deficient mice compared with wild-type mice that were challenged with ragweed, although this did not reach statistical significance (0.3 ± 0.3 adherent eosinophils/250 µm blood vessel length in ragweed-challenged TNF-R55-deficient mice versus 1.0 ± 0.4 adherent eosinophils/250 µm blood vessel length in ragweed-challenged wild-type mice; P = 0.10; n = 3) (Figure 7d).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have demonstrated that mice deficient in either TNF-R55/75 or TNF-R55 exhibit reduced BAL and peritoneal eosinophil recruitment compared with wild-type mice when challenged with allergen. Intravital videomicroscopy studies demonstrate that eosinophils exhibit both reduced rolling and firm adhesion to allergen-challenged endothelium in TNF-R55/75- and TNF-R55-deficient mice in vivo. This suggests that TNF released at sites of allergic inflammation in the lung may contribute to the recruitment of eosinophils during episodes of allergen-induced asthma through an indirect effect on eosinophils mediated by TNF upregulating endothelial cell adhesion molecules that bind circulating eosinophils. The absence of TNF-R type I (p55) on endothelial cells in TNF-R55/75- and TNF-R55-deficient mice would result in a reduced expression of endothelial adhesion molecules (16, 17) in response to TNF released at sites of allergen challenge and thus account for the reduced eosinophil rolling and adhesion we have noted. The reduced eosinophil recruitment we have observed both in the lung as well as in the peritoneal cavity of TNF-R55/75-deficient mice after allergen challenge is thus most likely a consequence of the reduced eosinophil rolling and adhesion to endothelium. Our study cannot exclude that reduced T helper (Th) 2 lymphocyte trafficking also contributed to the reduced eosinophil trafficking noted. However, in this study we have only evaluated eosinophil trafficking and similar studies with Th2 lymphocytes would need to be performed to characterize their trafficking patterns in TNF-R-deficient mice.

The importance of cytokines/mediators other than TNF to eosinophil adhesion to endothelium in vivo is suggested in this study from the fact that inhibition of eosinophil adhesion and recruitment is not complete in TNF-R55/75- and TNF-R55-deficient mice. The fact that allergen challenge induces rolling and adhesion of eosinophils in TNF-R55/75-deficient mice (compared with diluent challenge of TNF-R55/75-deficient mice) also suggests a non-TNF-mediated mechanism of eosinophil adhesion to endothelium. Nevertheless, the absence of TNF receptors significantly reduces eosinophil rolling, adhesion to endothelium, and tissue recruitment of eosinophils. In vitro cytokines such as IL-1 and TNF induce human umbilical vein endothelial cells to express several adhesion molecules, including VCAM-1, ICAM-1, E-selectin, and in the mouse, P-selectin) (29). In contrast, cytokines such as IL-4 induce the expression of a more restricted profile of endothelial cell adhesion molecules (IL-4 induces VCAM-1 but not ICAM-1 or E-selectin) (30). In vivo, specific endothelial-expressed adhesion molecules such as P-selectin (25, 32) and VCAM-1 (25, 33) are important to the initial eosinophil rolling on inflamed endothelium, whereas endothelial expressed adhesion molecules such as ICAM-1 (25, 33) and VCAM-1 (25, 33) are important to the subsequent eosinophil firm adhesion to endothelium. The availability of cytokine-deficient mice has allowed the investigation of the relative importance of individual cytokines to eosinophil recruitment after allergen challenge. Previous studies using cytokine-deficient mice other than TNF-R55/75- or TNF-R55-deficient mice (i.e., IL-1R type I- and IL-4- deficient mice challenged with aerosolized allergen) (22, 34) have also demonstrated reduced eosinophil recruitment to the lung, suggesting that several cytokines in addition to TNF contribute to eosinophil adhesion to endothelium in vivo.

Our study suggests that TNF was released at sites of allergic inflammation signals through endothelial-expressed TNF-R to promote eosinophil rolling and adhesion. As rolling, adhesion, and transmigration of eosinophils across endothelium are sequential steps, interruption of the adhesion cascade at an early step in TNF-R55/75- and TNF-R55- deficient mice reduces eosinophilic tissue inflammation. The potential for therapeutically targeting TNF in asthma is suggested from studies in a Schistosoma mansoni parasite model of eosinophilic inflammation in which parasitized mice treated with a soluble TNF- receptor linked to an Fc antibody molecule had significantly less eosinophilic inflammation compared with control mice (35). However, targeting TNF would not selectively inhibit eosinophil recruitment but would also inhibit recruitment of other circulating leukocytes (in addition to eosinophils) using the same profile of endothelial adhesion receptors induced by TNF (35). Indeed, the importance of TNF to neutrophil recruitment is suggested from studies of neutrophil recruitment to the lung after intranasal administration of Micropolyspora faeni antigen in mice deficient in the TNF-RI, which have shown that neutrophil recruitment to the lung is TNF- dependent (20).

Overall, this study suggests that TNF p55 receptors on endothelial cells play an important role in upregulating endothelial cell adhesion molecule expression at sites of allergen challenge as evidenced from our studies demonstrating reduced eosinophil rolling and adhesion in the allergen challenged mouse mesentery. Although TNF is neither a selective regulator of eosinophil recruitment nor the only cytokine regulating adhesion molecule expression by endothelium, this study nevertheless underscores the importance of TNF as one key mediator of the eosinophilic inflammatory response associated with allergic inflammation.