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(Journal of Leukocyte Biology. 2002;72:1122-1132.)
© 2002 by Society for Leukocyte Biology

Acute mast cell-dependent neutrophil recruitment in the skin is mediated by KC and LFA-1: inhibitory mechanisms of dexamethasone

Rene Schramm*, Thilo Schaefer{dagger}, Michael D. Menger{dagger} and Henrik Thorlacius*

* Department of Surgery, Malmö University Hospital, Sweden; and
{dagger} Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany

Correspondence: Henrik Thorlacius, Department of Surgery, University Hospital Malmö, SE-20502 Malmö, Sweden. E-mail: henrikthorlacius{at}hotmail.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study examined adhesive and signaling pathways and anti-inflammatory mechanisms of dexamethasone in acute mast cell-dependent neutrophil recruitment in the skin in mice. Mast cell activation dose- and time-dependently triggered influx of predominately neutrophils and secretion of cytokine-induced neutrophil chemoattractant (KC). Neutralization of KC attenuated neutrophil recruitment upon mast cell activation. Mast cell activation- and KC-induced neutrophil responses were significantly decreased in lymphocyte function-associated antigen-1 (LFA-1)-deficient mice. Dexamethasone inhibited neutrophil accumulation elicited by mast cell activation. It is interesting that dexamethasone significantly reduced the mast cell-dependent secretion of KC, whereas neutrophil recruitment induced by exogenous KC was insensitive to dexamethasone treatment. Thus, KC is a fundamental mediator of neutrophil recruitment in acute mast cell-dependent skin inflammation, and mast cell activation- and KC-induced neutrophil responses are LFA-1-dependent. Moreover, dexamethasone inhibits mast cell-regulated skin infiltration of neutrophils mainly by attenuating KC secretion. Thus, this study elucidates important interactions between chemokines and adhesion molecules in mast cell-dependent neutrophil recruitment and provides new insight into mechanisms of dexamethasone in skin inflammation.

Key Words: leukocyte • chemokines • intravital microscopy • glucocorticoids • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue mast cells have the capacity to initiate, amplify, and coordinate leukocyte recruitment and thus, play a key role in numerous inflammatory diseases including immediate and delayed hypersensitivity reactions in the gut, airways, and skin [1 2 3 4 ]. Leukocyte accumulation is a multistep process, which is orchestrated by specific chemotactic substances and adhesion molecules [5 ]. It is generally held that endothelial selectins(P- and E-selectin) support rolling as a precondition for the subsequent firm adhesion and tissue infiltration of leukocytes [6 ]. ß1- and ß2-integrins mediate stationary adhesion to the endothelium and extravascular migration of leukocytes [7 8 9 10 ]. Indeed, there is a growing body of evidence suggesting a dominant role for P-selectin in acute inflammatory neutrophil recruitment in general, and it was shown that P-selectin mediates leukocyte rolling in response to mast cell activation in the rat mesentery [11 , 12 ]. The functional significance of ß2-integrins in inflammatory leukocyte recruitment is illustrated by the leukocyte adhesion deficiency syndrome-1. A mutation of the gene encoding for CD18 results in a complete lack of all subtypes of ß2-integrins (CD11a-c/CD18), disabling neutrophil extravasation and thus, increasing susceptibility to recurrent bacterial infections [13 ]. Organ- and/or stimulus-dependent differences may help to explain controversial findings on the individual contribution of the two dominating ß2-integrins, lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) and Mac-1 (CD11b/CD18), to tissue accumulation of inflammatory cells [14 15 16 17 18 19 ]. However, a considerable amount of data suggests that LFA-1 is relatively more important in supporting firm adhesion of leukocytes to endothelial cells [9 , 20 , 21 ], whereas the role of LFA-1 in mast cell-dependent adhesion and recruitment of neutrophils remains to be defined.

Activation and trafficking of inflammatory cells are regulated by specific chemokines [6 ]. Although monocytes and lymphocytes are mainly reactive to CC chemokines, neutrophils are predominately responsive to members of the CXC chemokine family. In the mouse, the two best-defined CXC chemokines are cytokine-induced neutrophil chemoattractant (KC) and macrophage inflammatory protein-2 (MIP-2) [6 , 22 , 23 ]. It is interesting that it has recently been reported that T cell-mediated, delayed hypersensitivity reactions in the skin are dependent on mast cell-derived MIP-2 [4 ]. However, the expression and function of CXC chemokines in acute mast cell-dependent neutrophil infiltration in the skin are not known.

Glucocorticoids exert powerful anti-inflammatory effects and are important in the treatment of acute and chronic inflammatory diseases in the skin. Numerous studies have shown that the synthetic glucocorticoid dexamethasone attenuates dermal infiltration of leukocytes [24 , 25 ]. However, the detailed mechanisms of action of dexamethasone in mast cell-dependent acute inflammation and neutrophil recruitment in the skin remain elusive. Although our previous results strengthen the concept of dexamethasone attenuating leukocyte recruitment in cytokine-activated tissues by down-regulation of chemokine secretion [25 , 26 ], the literature provides contradictory data on whether glucocorticoids affect acute mast cell-dependent inflammatory reactions at the level of chemokine production and/or whether they directly interfere with leukocyte-endothelium interactions in the skin microcirculation. For example, one study has reported that mast cell-dependent synthesis of interleukin-8 (IL-8), a human homologue of murine MIP-2, is sensitive to dexamethasone [27 ], whereas others could not detect an inhibitory action of dexamethasone on IL-8 expression in mast cells [28 ].

Based on the considerations above, the objective of this study was to define the molecular mechanisms and kinetics of neutrophil recruitment and chemokine secretion and function in vivo using two models of cutaneous mast cell-dependent inflammation and to discern anti-inflammatory mechanisms of action of dexamethasone on neutrophil recruitment as well as chemokine secretion and function in acute mast cell-dependent inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male C57/Bl6, LFA-1-deficient (ref. [29 ]; a gift from Tak Mak, Department of Immunology, University of Toronto, Canada), tumor necrosis factor {alpha} (TNF-{alpha})-deficient (Jackson Laboratory, Bar Harbor, ME), and mast cell-deficient mice (WBB6F1; Jackson Laboratory), weighing between 22 and 25 g, were used. Mice were maintained in 12-h dark and 12-h light cycles and were given standard pellet food and water ad libitum. General anesthesia was achieved via intraperitoneal (i.p.) injection of 7.5 mg ketamine hydrochloride and 2.5 mg xylazine per 100 mg body weight. Dexamethasone (1 or 10 mg/kg; Decadron®, MSD, Netherlands) was given i.p., 2 h before local skin challenge. At the end of each experiment, a peripheral blood sample was taken from the tail artery for systemic differential leukocyte counts using a hematocytometer. All experiments were approved by the local ethics committee.

Air pouches
On days 0 and 3, 2.5 mL sterile air was inflated under the dorsal skin to raise subcutaneous (s.c.) air pouches as described before [25 , 26 , 30 ]. On day 6, indicated doses of the specific mast cell secretagogue compound 48/80 (CMP48/80; Sigma Chemical Co., St. Louis, MO), recombinant murine (rm)KC (0.5 µg; R&D Systems Europe, Ltd., Abingdon, Oxon, UK), rmMIP-2 (0.5 µg; R&D Systems Europe), or recombinant rat TNF-{alpha} (0.1 µg; R&D Systems Europe), dissolved in 1 ml phosphate-buffered saline (PBS), were injected into the air pouch cavities. Pouch exudates were harvested at indicated time points under anesthesia by washing the s.c. cavities three times with ice-cold PBS (1-2-2 ml) containing 3 mM EDTA. Harvested air pouch fluids were centrifuged at 1200 rpm (4°C) for 10 min, and the cell pellets were resuspended in 500 µl PBS. Total numbers of leukocytes per pouch were calculated from differential cell counts of 100 µl resuspended cells diluted v/v in Türk’s solution (crystal violet 0.01% w/v in acetic acid 3%) using a hematocytometer. Recruited cells were defined as polymorphonuclear (PMNL) or monomorphonuclear (MNL) leukocytes. Reduced leukocyte recruitment may be underestimated in animals with an increased systemic leukocyte count [31 ]. Thus, we calculated the recruitment efficiency in the LFA-1-deficient mice, which has previously been applied in these animals [9 , 21 ]. Recruitment efficiency is defined as the number of extravasated PMNLs in the pouch divided by the number of circulating PMNLs per nl blood. The importance of CXC chemokines and TNF-{alpha} in CMP48/80-induced cutaneous inflammation was studied by injection of a monoclonal antibody (mAb) directed against mMIP-2 [10 µg, rat immunoglobulin G (IgG) clone 40605.111; R&D Systems Europe], KC (10 µg, rat IgG clone 48415.111; R&D Systems Europe), or TNF-{alpha} (20 µg, rat IgG clone MP6-XT22; PharMingen, San Diego, CA) concomitantly with CMP48/80 into air pouches, and exudates were harvested and analyzed 4 h later. The role of LFA-1 in KC-induced neutrophil recruitment was determined in LFA-1-deficient mice and by use of mAb in LFA-1-expressing mice. Thus, mice were pretreated intravenously (i.v.) with an antibody against LFA-1 (M17/4.4.11.9, 100 µg/mouse, rat IgG; American Type Culture Collection, Manassas, VA) or a control antibody (9-A2, 100 µg/mouse, rat IgG; Bio-Express, West Lebanon, NH).

Intravital fluorescence microscopy in skin-fold chambers
For subsequent intravital fluorescence microscopy, a plexiglass chamber was inserted under the dorsal skin as described earlier [32 ]. Briefly, after i.p. anesthesia, an incision was made at the base of the back, and the dorsal skin was gently separated to allow nontraumatic insertion of the chamber. Subsequently, the chamber was fixed with one suture, and one layer of the skin was completely removed in a circular area of 15–20 mm in diameter. The remaining layer consisting of epidermis, s.c. tissue, and a thin striated skin muscle was covered with a coverslip. The animals tolerated the dorsal skin-fold chambers well and showed no signs of discomfort. In particular, there was no effect on sleeping or feeding habits. A recovery period of at least 48 h was allowed before intravital fluorescence microscopy of the skin microvasculature was performed. CMP48/80 (1 µg), dissolved in 100 µl PBS, or PBS alone was applied into the chamber by temporarily removing and replacing the coverglass, and 3 h later, leukocyte-endothelium interactions were evaluated analyzing three to five randomly chosen segments of different venules. Intravital microscopy was performed using an Olympus microscope (BX50WI) equipped with a water immersion lens (x40/NA 0.75). The microscopic images of the skin microvasculature were televised using a charge-coupled device videocamera (FK6990 Cohu, Pieper GmbH, Schwerte, Germany) and were recorded on videotape (Panasonic SVT-S3000 S-VHS recorder) for subsequent off-line analysis. After positioning under the microscope, a 10-min equilibration period preceded quantitative measurements. Analysis of leukocyte rolling and adhesion was made in postcapillary skin venules (25–40 µm) with stable resting blood flow. Rolling leukocyte flux was determined at indicated time points by counting the number of rolling leukocytes passing a reference point within 30 s and is given as cells/min. The number of firmly adherent leukocytes to the endothelium (stationary for >30 s) was counted along 150-µm venular segments and is given as cells/mm. For contrast enhancement of intravascular perfusion and in vivo labeling of leukocytes, the fluorescent dyes fluorescein isothiocyanate (FITC)-dextran (0.05 ml, MW 150,000, 5 mg/ml; Sigma Chemical Co.) and rhodamine 6G (0.1 ml, MW 479, 0.5 mg/ml; Sigma Chemical Co.) were injected into the right jugular vein 5–10 min before microscopic observations. Blood flow velocity and shear rate were measured and calculated using a video-assisted image analysis program (CapImage, Ingenieurbüro Zeintl, Heidelberg, Germany). To define the role of the endothelial selectins in leukocyte-endothelium interactions following mast cell activation, 40 µg mAb directed against endothelial P (RB40.34, rat IgG; PharMingen)- or E-selectin (10E9.6, rat IgG; PharMingen) or a control mAb (R3–34, rat IgG; PharMingen) were injected into the right jugular vein immediately before administration of CMP48/80 into the skin-fold chamber tissue.

Flow cytometry
Blood samples from C57/Bl6 and LFA-1-deficient mice were drawn from the abdominal aorta into EDTA tubes. Blood (50 µl) was incubated with a FITC-labeled rat (Lewis) anti-mouse CD11a mAb (M17/4, PharMingen) or a FITC-labeled isotype-matched control mAb (R3-34, PharMingen). To analyze the effect of dexamethasone on CXC chemokine-induced up-regulation of CD18 on neutrophils, C57/Bl6 mice were pretreated with dexamethasone (10 mg/kg) or PBS for 2 h. Blood samples were incubated with PBS or KC together with a FITC-labeled rat anti-mouse CD18 mAb (C71/16; PharMingen) or a control mAb. After incubation for 30 min at 4°C, the red blood cells were lysed (1 ml of NH4Cl EDTA for 5 min) and washed (1500 rpm) for 5 min. The cell pellet was resuspended with 1.0 ml PBS and put on ice until analysis, which was performed within 30 min. Neutrophils were gated based on forward- and side-scatter characteristics.

Reverse transcription-polymerase chain reaction (RT-PCR)
Neutrophils were freshly isolated from bone marrow of wild-type and CD11a gene-targeted mice. The bone marrow was flushed aseptically out of the femurs and humeri bones with ice-cold PBS, and neutrophils were then isolated by using Ficoll-Paque research grade (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The purity of bone marrow neutrophils was more than 70% as assessed by differential counting after staining with Türk’s solution using a hematocytometer. Total RNA was extracted from bone marrow neutrophils using RNeasy Mini-kit (Qiagen GmbH, Hilden, Germany) and was treated with RNase-free DNase (Amersham Pharmacia Biotech, Sollentuna, Sweden) to remove potential genomic DNA contaminants according to the manufacturer’s handbook. RNA concentrations were determined by measuring the absorbance at 260 nm spectrophotometrically. RT-PCR was performed with the SuperScript One-Step RT-PCR system (Gibco-BRL, Life Technologies, Grand Island, NY). Each reaction contained 250 ng total RNA as a template and 0.2 µM each primer, in a final volume of 50 µl. Mouse ß-actin served as an internal control gene. The RT-PCR profile was one cycle of cDNA synthesis at 50°C for 30 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min and one cycle of final extension at 72°C for 10 min. After RT-PCR, aliquots of the RT-PCR products were separated on a 2% agarose gel containing ethidium bromide and were photographed. The primers sequences were as follows: CD11a (f) 5'-AGA TCG AGT CCG GAC CCA CAG-3'; CD11a (r) 5'-GGC AGT GAT AGA GGC CTC CCG-3'; ß-actin (f) 5'-ATG TTT GAG ACC TTC AAC ACC-3'; ß-actin (r) 5'-TCT CCA GGG AGG AAG AGG AT-3'.

Enzyme-linked immunosorbent assay (ELISA)
Harvested air pouch exudates were centrifuged, and levels of immunoreactive MIP-2 and KC protein were determined in supernatants by means of double-antibody-specific Quantikine ELISA kits using rmMIP-2 and KC as standard (R&D Systems Europe). TNF-{alpha} levels were quantified by two distinct ELISA kits with rmTNF-{alpha} as standard (R&D Systems Europe and Endogen, Cambridge, MA). The minimal detectable protein concentrations are <0.5 pg/ml in these kits.

Experiments in gene-targeted mice
In separate experiments, the role of LFA-1 and TNF-{alpha} in mast cell-dependent neutrophil recruitment was studied in gene-targeted animals. As described above, leukocyte-endothelium interactions, i.e., rolling and adhesion, were studied in the dorsal skin-fold chamber, and leukocyte recruitment was assessed in air pouches.

Histology
To analyze the effect of dexamethasone on skin mast cell numbers, air pouch tissue was harvested after 6 h treatment with dexamethasone and processed for paraffin-embedding. Skin tissue specimens were also harvested from air pouches of LFA-1-deficient animals and were prepared for histological analysis. Subsequently, sections of 6 µm were stained with toluidine blue, and mast cells were identified and counted in 40 high-power fields per specimen using normal light microscopy (40x magnification).

Statistics
Data are given as mean values ± SEM, and n represents the number of animals per experimental group. Statistical differences between treatments were assessed by one-way ANOVA, followed by the Dunnet post-hoc test (SigmaStat 4.0, Jandel Scientific, Chicago, IL). Differences were considered to be significant at values of probability <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mast cell activation induces acute inflammation in the skin
Injection of CMP48/80 evoked an acute inflammatory reaction in the skin characterized by a dose-dependent elevation of leukocyte numbers in air pouch exudates at 4 h after challenge (Fig. 1A ; n=8–19). Differential counts revealed that PMNLs comprised more than 70% of the extravasated cells, suggesting that the response was mainly neutrophilic in nature. The greatest leukocyte response was observed after stimulation with 1 µg CMP48/80; i.e., extravascular PMNLs and MNLs were 502 ± 48 and 195 ± 21 x 103, respectively (Fig. 1A) . Importantly, we confirmed that 1 µg CMP48/80 caused a mast cell-dependent response; i.e., the number of extravascular leukocytes in the skin of mast cell-deficient animals (270±48x103 PMNLs/pouch) was significantly lower than that in wild-type mice (P<0.05 vs. CMP48/80-treated wild-type mice, n=5–19). Moreover, histological analysis revealed that tissue specimens harvested from WBB6F1 mice were completely mast cell-deficient (data not shown). Thus, based on the above findings, all subsequent air pouch experiments were performed using 1 µg CMP48/80. Moreover, this dose of CMP48/80 caused a time-dependent leukocyte response, peaking at 4 h after stimulation and returned to baseline values between 12 h and 24 h (Fig. 1B) .



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  		Figure 1. Leukocyte recruitment after mast cell activation with CMP48/80. (A) The dose-dependent effect was studied at 4 h after challenge with CMP48/80. (B) The time-dependent effect was evaluated using 1 µg/ml CMP48/80. Differential counts of leukocytes in harvested air pouch exudates were assessed after staining with Türk’s solution using a Bürker chamber. Data are mean values ± SEM; *P < 0.05 versus 0 µg CMP48/80, n = 8–19; #P < 0.05 versus 0 h, n = 7–19.

 
CXC chemokines and TNF-{alpha} in acute mast cell-dependent inflammation
We observed that mast cell activation triggered KC secretion in air pouches (Fig. 2 ). In fact, the levels of KC in the air pouch exudates increased time-dependently after challenge with CMP48/80 (Fig. 2) . Maximum expression of KC was detected after 1 h (195±61 pg/pouch) and returned to baseline values between 4 h and 24 h (Fig. 2) . Conversely, MIP-2 was undetectable up to 24 h after mast cell activation (Fig. 2) . It is interesting that immunoneutralization of KC by local treatment with an anti-KC mAb significantly attenuated the CMP48/80-induced s.c. accumulation of neutrophils by nearly 64% (Fig. 3 ). In contrast, administration of a mAb directed against MIP-2 had no effect on the neutrophil extravasation in response to mast cell activation (Fig. 3) . Neither mAb significantly altered the CMP48/80-induced extravasation of MNLs (Fig. 3) . In addition, mast cell-dependent leukocyte accumulation was found to be insensitive to local administration of mAb against TNF-{alpha}, and the leukocyte response was intact in TNF-{alpha}-deficient animals (data not shown). Indeed, TNF-{alpha} was not detected in pouch exudates of wild-type mice after mast cell degranulation (Table 1 ).



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  		Figure 2. Time-dependent secretion of CXC chemokines in murine air pouch exudates in response to 1 µg CMP48/80. Exudates were harvested, and supernatants were analyzed for KC (solid circles) and MIP-2 protein (open circles) by means of ELISA. Data are mean values ± SEM; *P < 0.05 versus 0 h, n = 6–12.

 


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  		Figure 3. Role of CXC chemokines in CMP48/80-induced leukocyte recruitment. mAb against MIP-2 and KC were given locally, concomitant with the injection of 1 µg CMP48/80 into air pouches, and exudates were harvested and analyzed at 4 h after challenge. PBS alone served as negative control. Data are mean values ± SEM; #P < 0.05 versus negative controls, n = 10–19; *P < 0.05 versus anti-MIP-2 treatment, n = 7.

 

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  		Table 1. Effects of Dexamethasone Treatment on KC Expression in Response to CMP48/80

 
Leukocyte-endothelium adhesive interactions elicited by mast cells
Intravital fluorescence microscopy of the dorsal skin-fold microcirculation revealed that leukocyte rolling in control mice and CMP48/80-treated animals was 16.3 ± 4.1 and 14.3 ± 3.7 cells/min, respectively (n=6–8). Injection of the mAb directed against endothelial P-selectin significantly reduced leukocyte rolling to 6.8 ± 1.5 cells/min at 3 h after challenge with CMP48/80 (Fig. 4A ), and the anti-E-selectin mAb had no effect (Fig. 4A) . Moreover, mast cell activation significantly increased leukocyte firm adhesion, i.e. 30.2 ± 4.2 and 60.3 ± 8.1 cells/mm at 3 h after challenge with PBS and CMP48/80, respectively (P<0.05 vs. PBS, n=8–9). Treatment with anti-P-selectin mAb significantly reduced CMP48/80-induced leukocyte firm adhesion; i.e., the number of firmly adherent leukocytes was reduced by 58% down to 20.3 ± 3.9 cells/mm (Fig. 4B) . Administration of the anti-E-selectin mAb had no effect on mast cell-dependent leukocyte firm adhesion (Fig. 4B) .



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  		Figure 4. Leukocyte rolling and firm adhesion in the dorsal skin-fold microcirculation after local administration of CMP48/80. mAb directed against P- and E-selectin and a control mAb were given i.v. just before challenge with CMP48/80. The numbers of (A) rolling and (B) firmly adherent leukocytes were determined by the use of intravital fluorescence microscopy performed at 3 h after local challenge with 1 µg CMP48/80. Data are mean values ± SEM; *P < 0.05 versus control mAb, n = 6–9.

 
LFA-1 mediates mast cell-dependent neutrophil recruitment
Flow cytometric analyses revealed that surface expression of LFA-1 was reduced by more than 95% on neutrophils in gene-targeted mice compared with wild types (data not shown). Additionally, we found no detectable CD11a mRNA levels in bone marrow neutrophils harvested from LFA-1-deficient animals, indicating a complete deletion of the CD11a gene (data not shown). Leukocyte rolling at 3 h after mast cell activation was intact in LFA-1-deficient animals (Fig. 5A ). In contrast, the number of firmly adherent leukocytes was significantly reduced by more than 53% in LFA-1 mutant mice (Fig. 5B) . Moreover, we found that pouch extravasation of neutrophils in response to mast cell activation was decreased by 49% in LFA-1-deficient animals (P<0.05 vs. wild types, n=5–19), suggesting an important role of LFA-1 in mast cell-dependent neutrophil recruitment in the skin.



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  		Figure 5. Importance of LFA-1 in CMP48/80-induced leukocyte recruitment. (A) Leukocyte rolling and (B) firm adhesion in the dorsal skin-fold microcirculation of wild-type and LFA-1-deficient mice. Intravital fluorescence microscopy was performed at 3 h after local administration of 1 µg CMP48/80. Data are mean values ± SEM; *P < 0.05 versus wild type, n = 5–9.

 
Dexamethasone inhibits mast cell-regulated skin inflammation
Systemic treatment with dexamethasone decreased the CMP48/80-induced leukocyte accumulation in murine air pouches (Fig. 6 ). In fact, pretreatment with 10 but not with 1 mg/kg dexamethasone significantly decreased the numbers of extravasated neutrophils by 41% at 4 h after mast cell activation (Fig. 6) . Dexamethasone did not interfere with s.c. leukocyte accumulation at 1 h after mast cell activation (data not shown). CMP48/80-induced MNL trafficking was not significantly altered by either dose of dexamethasone at any time point (Fig. 6) . Notably, we found that KC expression in the air pouches elicited by mast cell activation was significantly decreased (47% reduction) by pretreatment with 10 mg/kg but not 1 mg/kg dexamethasone (Table 1) . Intravital microscopy of the skin microcirculation revealed that treatment with 10 mg/kg dexamethasone did not alter leukocyte rolling in CMP48/80-treated mice (Fig. 7A ). Conversely, systemical treatment with dexamethasone markedly attenuated CMP48/80-induced leukocyte firm adhesion by 73% (Fig. 7B) . Similar to the high dose, treatment with 1 mg/kg dexamethasone reduced only leukocyte adhesion but not rolling. Histological analysis revealed that neither dose of dexamethasone decreased skin mast cell numbers, and there was no difference in the number of skin mast cells between wild-type and LFA-1-deficient animals (data not shown).



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  		Figure 6. Effects of dexamethasone on CMP48/80-induced s.c. leukocyte accumulation. Mice were treated i.p. with or without dexamethasone (1 and 10 mg/kg) 2 h before local challenge. Air pouch exudates were harvested and analyzed 4 h after stimulation with 1 µg CMP48/80. Data are mean values ± SEM; *P < 0.05 versus control, n = 9–19.

 


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  		Figure 7. Effects of dexamethasone on mast cell-dependent leukocyte-endothelium interactions. (A) Leukocyte rolling and (B) firm adhesion were analyzed in the dorsal skin-fold microcirculation after local administration of CMP48/80 by use of intravital microscopy. Mice were treated i.p. with or without dexamethasone (10 mg/kg) 2 h before local challenge with 1 µg CMP48/80. Negative controls were locally treated with PBS alone. Data are mean values ± SEM; #P < 0.05 versus negative controls, n = 6–8; *P < 0.05 versus CMP48/80-treated controls, n = 6–7.

 
CXC chemokine-induced neutrophil recruitment
Injection of KC into air pouches caused a dramatic and neutrophil-dominated (>81%) leukocyte response (Fig. 8 ). Four hours after stimulation with 0.5 µg KC, an average amount of 1104 ± 200 x 103 neutrophils was harvested from the air pouch cavities (Fig. 8) . It was found that KC-provoked recruitment was supported by LFA-1 (Table 2 ). In fact, recruitment efficiency [9 , 21 , 31 ] after challenge with KC was 207 ± 48 and 2850 ± 820 PMNLs/pouch per 106 PMNLs/ml blood in LFA-1-deficient and wild-type mice, respectively (Table 2) . These findings were validated by use of a mAb against LFA-1. Thus, the number of recruited PMNLs was 1826 ± 378 x 103 and 598 ± 254 x 103 PMNLs/pouch in mice receiving a control antibody and the anti-LFA-1 antibody, respectively (Table 2) . It is interesting that pretreatment with 10 mg/kg dexamethasone was not capable to significantly decrease the KC-induced dermal neutrophil accumulation; i.e., the number of extravasated neutrophils was 952 ± 186 x 103 (Fig. 8) . Furthermore, KC increased CD18 surface expression on neutrophils twofold. This increase was not affected by pretreatment with dexamethasone (10 mg/kg). In fact, after incubation with KC, mean fluorescence intensity on neutrophils harvested from PBS- and dexamethasone-treated wild-type mice was 46.6 ± 2.0 (n=3) and 45.5 ± 2.7 (n=3), respectively. In contrast to the neutrophil response elicited by KC, we observed that treatment with 10 mg/kg dexamethasone significantly reduced MIP-2-provoked neutrophil recruitment by more than 79% (P<0.05 vs. PBS, n=6). Additionally and in line with our previous findings [25 ], injection of 0.1 µg TNF-{alpha} induced a marked secretion of MIP-2 in murine air pouches at 1 h after challenge, i.e., 998 ± 212 pg/pouch (n=3).



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  		Figure 8. Effects of dexamethasone on KC-induced s.c. leukocyte accumulation. Dexamethasone (10 mg/kg) or PBS alone was given i.p. 2 h before local challenge with 0.5 µg KC, and air pouch exudates were harvested and analyzed after 4 h after local challenge. Data are mean values ± SEM; n = 7.

 

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  		Table 2. Role of LFA-1 in KC-Induced Neutrophil Recruitment

 
Hemodynamic parameters and blood cell analyses
Blood-flow velocities and shear rates in microvessels studied did not differ between the experimental groups (Table 3 ). The effects of dexamethasone on levels of circulating PMNLs and MNLs are shown in Table 4 and Table 5 .


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  		Table 3. Hemodynamic Parameters

 

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  		Table 4. Systemic Leukocyte Differential Counts in Animals Used for Intravital Microscopy

 

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  		Table 5. Systemic Leukocyte Differential Counts in Animals Used for Air Pouch Experiments

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study describes important signaling and adhesive mechanisms behind neutrophil recruitment and a key target of dexamethasone in acute mast cell-dependent inflammation in the skin. Our data demonstrate that activation of cutaneous mast cells induces massive secretion of KC, which turned out to be instrumental in mast cell-mediated infiltration of neutrophils. Moreover, these findings show that leukocyte rolling is mainly supported by P-selectin, whereas firm venular adhesion and dermal accumulation of neutrophils are predominately mediated by LFA-1. The observation that KC-induced neutrophil recruitment was dependent on LFA-1 is coherent with our concept that KC is a key mediator of the neutrophil response to mast cell activation. Moreover, we found that dexamethasone potently inhibits neutrophil accumulation elicited by mast cells. Indeed, KC expression on mast cell activation was markedly reduced by dexamethasone, whereas neutrophil recruitment triggered by KC was found to be insensitive to dexamethasone, suggesting that glucocorticoids primarily attenuate neutrophil recruitment in mast cell-dependent acute inflammation via inhibition of KC secretion.

Leukocyte recruitment to sites of inflammation is coordinated by a complex interplay between chemokines and adhesion molecules. Mast cells are considered to play an important role in early and late phases of pathological inflammation as an intermediate cellular source of inflammatory mediators [3 , 4 , 33 34 35 ]. Previous studies have forwarded the importance of numerous, different mast cell-derived mediators, which may be related to the heterogeneity of mast cell subpopulations in different anatomic localizations [36 ]. This underlines the value of studying individual tissues to understand organ-specific characteristics of mast cell-dependent recruitment of inflammatory cells. A recent study by Biedermann et al. [4 ] described in detail the role of mast cells in the development of T cell-mediated, delayed-type hypersensitivity reactions in the skin. However, the signaling and adhesive mechanisms behind dermal accumulation of neutrophils in mast cell-mediated acute inflammation are not known. In the present study, we studied acute mast cell-dependent neutrophil recruitment using CMP48/80, which is a specific mast-cell degranulator [37 38 39 ]. In fact, administration of CMP48/80 provoked a dose- and time-dependent infiltration of neutrophils in wild-type animals, whereas no proinflammatory effect was observed in mast cell-deficient mice, suggesting that CMP48/80 indeed activated mast cells. It is interesting that we found that specific activation of cutaneous mast cells provoked a rapid and great secretion of KC. Moreover, administration of an anti-KC antibody markedly decreased neutrophil recruitment in response to mast cell activation. In contrast, we did not detect MIP-2 in pouch exudates, and neutrophil recruitment was not affected by immunoneutralization of MIP-2 in acute mast cell-dependent inflammation. These findings suggest that KC and not MIP-2 is a critical mediator of neutrophil accumulation in acute mast cell-dependent inflammation in the skin. Supporting this notion as a general concept in acute mast cell-dependent inflammation, two previous studies showed that mast cell-derived KC mediates acute neutrophil recruitment induced by IL-1ß and zymosan-activated plasma (a source of C5a) in the mesentery and peritoneal cavity, respectively [34 , 35 ]. Nonetheless, it must be mentioned in this context that it was recently reported that mast cell-derived MIP-2 regulates dermal accumulation of neutrophils in a delayed-type hypersensitivity reaction [4 ]. However, in relation to mast cell-dependent neutrophil recruitment, two important differences between that study and our present investigation are that Biedermann et al. [4 ] examined a T cell-mediated reaction (hapten-induced) and neutrophil responses at a late(r) time point (24 h). Considered collectively, it may be suggested that mast cell-dependent neutrophil infiltration is regulated by KC in the acute phase and by MIP-2 in the delayed phase of skin inflammation. Indeed, it is interesting to note that Biedermann et al. [4 ] only detected MIP-2 mRNA expression after the secondary hapten exposure, whereas KC mRNA was already present after primary sensitization. In line with this observation, we have previously shown that KC mRNA but not MIP-2 mRNA is expressed at baseline levels in the striated muscle of nontreated control mice [40 ]. The functional significance of such a baseline expression of KC mRNA is not known, but it may be speculated that this baseline KC mRNA is rapidly translated into a functional protein and thus constitutes an integral part of an efficient defense system in response to bacterial invasion.

Neutrophil recruitment in acute inflammation is not only promoted by chemokines but is also regulated by potent cytokines such as TNF-{alpha} [41 , 42 ]. For example, it has been shown that TNF-{alpha} induces CXC chemokine-dependent infiltration of neutrophils in the skin [25 , 30 ]. However, we did not detect TNF-{alpha} in pouch exudates by use of two different ELISA kits, including rmTNF-{alpha} as a positive and detectable control. Moreover, mast cell activation-induced neutrophil accumulation was not reduced by pretreatment with an antibody directed against mTNF-{alpha}, and furthermore, the neutrophil response to mast-cell activation was intact in TNF-{alpha}-deficient mice. Thus, these lines of evidence clearly demonstrate that TNF-{alpha} is not an important mediator in the recruitment of neutrophils during acute mast cell-mediated skin inflammation. This conclusion is also supported by our data, showing on one hand that MIP-2 was undetectable after mast cell activation and conversely, that TNF-{alpha} markedly increased MIP-2 levels in the skin, suggesting that TNF-{alpha} was not present in the skin at significant concentrations after mast cell activation. It is interesting to note that Biedermann et al. [4 ] reported a key role of mast cell-derived TNF-{alpha} in the neutrophil accumulation during T cell-mediated, delayed-type hypersensitivity reactions, which adds another distinct difference in the mechanistic patterns of acute and late phases of mast cell-regulated skin infiltration of inflammatory cells.

Adhesion molecules on the surface of endothelial cells and circulating leukocytes facilitate adhesive interactions in the extravasation process of inflammatory cells [5 ]. Tissue inflammation is characterized by increased expression of endothelial selectins, i.e., P- and E-selectin [43 ]. Herein, it was found that administration of an anti-P-selectin antibody significantly reduced rolling and firm adhesion of leukocytes along the microvascular endothelium in response to mast cell activation. These findings suggest not only that leukocyte rolling is mediated by P-selectin but also that the rolling adhesive interaction is a precondition for the subsequent leukocyte firm adhesion in mast cell-dependent inflammation in the skin. This conclusion is supported by two previous studies showing a similar role of P-selectin in the rat mesentery [12 , 44 ]. Moreover, inhibition of E-selectin had no effect on mast cell activation-induced leukocyte-endothelium interactions in the skin. In this context, it is important to note that E-selectin may not primarily be involved in supporting the rolling adhesive interaction but rather may facilitate downstream events such as activation of rolling leukocytes [45 , 46 ]. This notion is also supported by a previous study, which demonstrated that immunoneutralization of E-selectin markedly reduced peritoneal recruitment of neutrophils but had no effect on TNF-{alpha}-induced leukocyte rolling [47 ].

Firm leukocyte adhesion to the vascular endothelium is mediated through ß2-integrins, preferably LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18). However, the literature on the relative importance of the individual ß2-integrins in leukocyte adhesion is complex and appears to vary depending on the type of inflammatory stimulus and the experimental model used [9 , 18 19 20 ]. The present study demonstrates for the first time that mast cell-regulated firm adhesion and tissue recruitment of neutrophils in the skin are critically dependent on LFA-1. In fact, the number of adherent and extravascular neutrophils was reduced by 53% and 49%, respectively. Importantly, we could document that LFA-1 was completely deleted at the mRNA and protein level. This important role of LFA-1 in mast cell-dependent inflammation is in line with earlier findings in these gene-targeted mice, showing that LFA-1 is a major adhesion molecule in supporting leukocyte adhesion triggered by TNF-{alpha} [9 , 48 ], ischemia-reperfusion (A. A. Riaz et al., unpublished data), and thioglyccolate [29 ]. Moreover, we found that KC-induced neutrophil recruitment was markedly decreased in LFA-1-deficient mice and by immunoneutralization of LFA-1 in wild-type animals, emphasizing the importance of LFA-1 in mediating neutrophil recruitment in the skin. In fact, the observation that mast cell-dependent and KC-provoked accumulation of neutrophils are mediated by LFA-1 lends further support to our concept that KC is a key component in mast cell-regulated inflammation in the skin. An interesting study by Inamura et al. [49 ] demonstrated that T cell-mediated degranulation of mast cells is dependent on LFA-1/intercellular adhesion molecule-1 interactions. Considered together with the present findings, it may be suggested that LFA-1 not only plays at least two different mechanistic roles in mast cell-dependent inflammation (i.e., T cell-mediated degranulation of mast cells and mast cell-dependent neutrophil adhesion) but may also be a critical molecule in acute and delayed-type skin reactions.

Glucocorticoids are potent anti-inflammatory agents with a broad spectrum of inhibitory actions on cells involved in inflammatory processes [50 ]. However, the anti-inflammatory mechanisms of dexamethasone on mast cells remain elusive, which is, at least partly, attributable to a previous lack of understanding the mechanisms of acute mast cell-dependent inflammation in the skin. The characterization of important signaling and adhesive pathways in neutrophil recruitment on mast cell activation in the present study permitted us to define potential key targets of dexamethasone, which may facilitate the development of more specific and safer drugs against inflammatory diseases in the skin. We found that dexamethasone reduced the number of adherent and extravascular neutrophils in response to mast cell activation in the skin. In fact, the reduction in firm adhesion and recruitment of neutrophils correlated very well to the decrease in KC secretion in dexamethasone-treated animals. Conversely, treatment with dexamethasone had no effect on KC-induced infiltration of neutrophils in the skin. It has been forwarded that dexamethasone may decrease inflammatory cell recruitment via down-regulation of CD18 on circulating leukocytes [51 52 53 ], although we and others [25 , 54 , 55 ] have not been able to confirm such findings. Herein, we also found that up-regulation of CD18 expression on neutrophils provoked by KC was insensitive to dexamethasone treatment. Knowing that mast cell-dependent and KC-induced neutrophil recruitment in the skin are mediated by LFA-1 (CD11a/CD18), it is suggested that CD18 is not an important target of glucocorticoids in acute mast cell-mediated inflammation. This conclusion is also supported by our present findings demonstrating that dexamethasone inhibits mast cell-dependent (inhibition of KC secretion) but not KC-induced neutrophil recruitment. Thus, considering that KC plays an important function in mast cell-dependent accumulation of neutrophils in the skin (this study), our data indicate that KC secretion is a key target of dexamethasone in acute inflammation triggered by mast cells. This notion is also in line with observations made by Tailor et al. [35 ]demonstrating that dexamethasone reduces the production of mast cell-derived KC in a model of acute inflammation in the rat mesentery. Based on the aforementioned findings, we predict that targeting KC synthesis may be effective in treating pathological inflammation in the skin regulated by mast cells.

In conclusion, this study demonstrates that KC is a fundamental mediator of neutrophil recruitment in acute mast cell-dependent inflammation in the skin. Moreover, we provide evidence that mast cell activation and KC-induced firm adhesion and tissue accumulation of neutrophils are dependent on LFA-1 function. One major target of dexamethasone in mast cell-regulated infiltration of neutrophil in the skin is the expression of KC, whereas the function of KC and expression of CD18 are insensitive to dexamethasone treatment. Thus, our novel findings define important signaling and adhesive pathways of neutrophil trafficking and clarify complex and critical anti-inflammatory mechanisms of dexamethasone in acute mast cell-dependent inflammation in the skin.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Swedish Medical Research Council (K2000–04P-13411–01A, K2002–73-X-14273–01A), Cancerfonden (4265-B99–01XAB), Crafoordska stiftelsen (20010968), Blaceflors stiftelse, Einar och Inga Nilssons stiftelse, Harald och Greta Jaenssons stiftelse, Greta och Johan Kocks stiftelser, Fröken Agnes Nilssons stiftelse, Franke and Margareta Bergqvists stiftelse för främjande av cancerforskning, Nanna Svartz stiftelse, Ruth och Richard Julins stiftelse, Svenska Läkaresällskapet (2001–907), Teggers stiftelse, Allmäna sjukhusets i Malmö stiftelse för bekämpande av cancer, MAS fonder, and Malmö University Hospital and Lund University.

Received June 8, 2002; revised September 18, 2002; accepted September 20, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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