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Intramuscular injection of hrRANTES causes mast cell recruitment and increased transcription of histidine decarboxylase in mice: lack of effects in genetically mast cell-deficient W/W^V mice

^a Immunology Division, University of Chieti School of Medicine, Chieti, Italy
^b Department of Pharmacology, Tufts University School of Medicine, Boston Massachusetts, USA

	ABSTRACT

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RANTES (regulated upon activation, normal T cell expressed and presumably secreted) and other chemoattractant proteins are members of the intercrine or chemokine family of proinflammatory basic polypeptides. RANTES is a prototype of the C-C chemokine subfamily that acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. However, the role of RANTES in white cells is still unclear. We report here that hrRANTES at 20 ng/50 µl in mice causes mast cell recruitment 4 h after intramuscular injection, an effect inhibited by anti-RANTES, as evidenced by 0.1% Toluidine blue, a specific dye for coloring mast cells. Injections of PBS (50 µl) vehicle (negative control) did not produce any appreciable inflammatory response, whereas injection of lipopolysaccharide 20 ng/50 µl (positive control) generated a marked inflammatory state. When RANTES was injected intramuscularly in genetically mast cell-deficient W/W^v mice, the inflammatory effect was not present. The RANTES injection sites were then excised and studied under an optical and electron microscope. A Northern blot analysis was performed using a probe that was prepared to detect mRNA encoding the histidine decarboxylase (HDC) gene on excised muscle tissue. We found that hrRANTES provoked generation of HDC mRNA from muscle tissue after 4 h. These effects were inhibited by an anti-RANTES antibody and were absent in genetically mast cell-deficient mice. The increasing number of mast cells in the RANTES injection sites led to an augmentation of histamine content compared to controls (PBS). The injection of hrRANTES 20 ng/20 µl into the sole of a rat paw confirmed the inflammatory and the mast cell recruitment potential of this chemokine. In these studies, hrRANTES injections in muscle tissue provided direct in vivo evidence that RANTES has a significant effect on mast cell recruitment and HDC mRNA generation.—Conti, P., Reale, M., Barbacane, R. C., Letourneau, R., Theoharides, T. C. Intramuscular injection of hrRANTES causes mast cell recruitment and increased transcription of histidine decarboxylase in mice: lack of effects in genetically mast cell-deficient W/W^V mice. FASEB J. 12, 1693–1700 (1998)

Key Words: chemokines • HDC • histamine • polysaccharide

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
HUMAN CHEMOKINES ARE small 7–10 kDa proteins that are subdivided in the CXC, CC, and C chemokine family of chemotactic cytokines for either neutrophils, mononuclear cells, basophils, eosinophils, or lymphocytes (1–3). Leukocyte adhesion and infiltration (4) into inflammatory tissue is mediated by chemotactic chemokines. Chemokines display three highly conserved cysteine amino acid residues. CC chemokines are clustered on human chromosome 17. A prototype of this subfamily includes RANTES (regulated upon activation, normal T cell expressed and presumably secreted),² which has selective chemoattractant properties in monocyte/macrophages, naive T cells (CD45RA+) and basophils rather than neutrophils (5–6). RANTES is reported to be chemotactic for basophilic cells in a rat skin model (7–9).

RANTES is a member of the ß intercrine subfamily reported to be a mediator in atopic pathologies characterized by inflammatory responses. Moreover, this chemokine is involved in the kinetics of allergen-induced transcription of mRNA in the skin of human atopic subjects, in the formation of eosinophil and monocytic intradermal inflammatory sites in the dog, and in basophilic recruitment in rat skin injection sites (10, 11). Basophils and mast cells are involved in the irritation of human allergic reactions by virtue of aggregation of immunoglobulin E (IgE) bound to high-affinity receptor (FRI) on their surface (12, 13), resulting in degranulation and release of mediators such as histamine, proteases, chemotactic factors, arachidonic acid metabolites, and cytokines (3, 14). Mature mast cells reside in connective tissues and accumulate in a variety of diseases where inflammation is mediated (15–17). However, the recruitment of basophils and mast cells by chemokines in inflammatory sites is not yet clearly understood (7, 18, 19).

Recently, it was reported that the two beta chemokines, RANTES and monocyte chemotactic protein 1 (MCP-1), induce mast cell migration without degranulation in extracellular matrices, an effect enhanced by IgE-dependent activation (20). However, other authors report that RANTES and MCP-1 are potent basophil agonists that induce rapid changes of cytosolic free calcium (Ca²⁺), concentration, and release of histamine and chemotaxis (21).

As depicted in this paper, we studied the effect of RANTES injected in muscle tissue sites on mast cell recruitment at the sites of injection in normal and genetically mast cell-deficient W/W^v mice. We also determined, at the mRNA level, the effect of RANTES on histidine decarboxylase (HDC) activation in normal and mast cell-deficient mice. HDC is the rate-limiting enzyme responsible for the generation of histamine from histidine. In these studies, the addition of polyclonal anti-RANTES always blocked the biological effects produced by exogenously administered human recombinant (hr)RANTES.

	MATERIALS AND METHODS

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Intraquadricept muscle injection of hrRANTES in normal and C57BL/6J-W^v/+ mice
Mice of a mutant genotype are useful in investigations of the origin of tissue mast cells and for analysis of mast cell biology. W/W^v mice express profound deficiency of mast cells (22–25). The number of mast cells in the tissue of WBB6F₁ (WB-W/+xC57BL/6-W^v/+) - W/W^v is less than 1% of the value for the congenic +/+ mice (22–25).

C57BL/6J-W^v/+ mice were obtained from Jackson Lab. (Bar Harbor, Maine). The W^v gene was maintained by repeated backcrosses of W^v/+ male mice to C57BL/6-+/+ female mice of an inbred colony. WB-W/+ mice were also obtained from Jackson Lab. and maintained by brother-sister mating. These mice were used in all experiments. Before intramuscular injection of the compounds or phosphate-buffered saline (PBS, vehicle), the mice were fasted 24 h with free access to water and kept in separate cages with a screen on the bottom to prevent coprophagy. The experimental animals used were homogeneous for weight and age and were raised under the same environmental and feeding conditions. Mice were anesthetized with ketamine HCl (80 mg/kg) (USP, Fort Dodge Lab. Inc., Iowa) plus xylazine HCl (10 mg/kg) in 0.99% NaCl volume in a 0.05 ml injection (Fermenta Animal Health Co., Kansas City, Mo.). The injection was made in the lower right quadrant of the abdomen. After 5 min, the quadriceps of the mice were shaved under anesthesia and injected with a 50 µl of RANTES, PBS (negative control) and lipopolysaccharide (LPS) from E. coli (positive control) serotype 0111: B4 TCA extract (Sigma, St. Louis, Mo.; catalog or stock no. L-4130). Four hours after the intramuscular injections, the animals were killed by inhalation of CO₂, followed by decapitation. The injected areas were enucleated and immersed in a fixative solution of 5% formaldehyde (Polyscience Inc., Warrington, Pa.) plus PBS for at least 10–15 min before use. Slides were prepared with sections of the tissue, stained with Toluidine blue (0.1% in 1% sodium borate for 2 min), and analyzed under an optic microscope (x10, x20, x40, and x100, Nikon Diaphore THD microscope, Japan). The mast cells were counted in the optic field using a grating size 5 x 5 mm.

Human recombinant (hr)RANTES (specific activity above 10 ng/ml, according to the manufacturer) was purchased from R&D systems (Minneapolis, Minn.). Preparation of hrRANTES contained less than 1 unit/mg of endotoxin protein as determined by the Limulus amebocyte lysate reaction (LPS levels were <100 pg/µg of protein and the protein preparations were inactive in the same dosage range in the mouse).

Anti-RANTES neutralizing antibody in normal mice
An anti-RANTES neutralizing antibody total goat IgG (R&D Systems; catalog no. AB278NA, lot no. DP06) was used at the concentration of 50 µg/mouse. The antibody was injected intravenously in the tail vein with a special Precision Glide needle (30G1/2, Becton Dickinson, Franklin Lakes, N.J.) in 100 µl PBS. After 30 min, RANTES was injected in the muscle tissue. The reconstituted antibody was used immediately.

Electron microscopy studies
Other tissue samples with injected compounds were removed for electron microscopy analysis (26). The samples were fixed for 1 h at room temperature with 3% glutaraldehyde in 0.1 M cacodylate-HCl buffer (pH 7.2). The tissues were then infiltrated with two 10 min changes of 100% propylene oxide, followed by overnight exposure to a 1:1 mixture of propylene oxide and DMP-30. The next day, the muscle tissues were embedded in Epon with DMP-30. Embedded tissues were placed in a 56°C oven to polymerize for 48 h. Thick and thin sections were cut on Sorvall MT-1 and MT-2B Ultramicrotomes equipped with glass and diamond knives, respectively. Sections (1000 Å) were picked up on 300 mesh copper grids and stained with both uranium and lead salts. The sections were then examined and photographed using a JEOL JEM-100s transmission electron microscope operated at an accelerating voltage of 80 kV.

Preparation of the HDC probe
We used a probe made from a reverse transcribed rat brain polyA+ RNA. Total cellular RNA was extracted from rat brains from New England Deaconess Hospital (Boston, Mass.). PolyA mRNA was purified according to Bruneau et al. (27). A sample of 2 µg polyA+ mRNA was reverse transcribed at 42°C for 40 min in a 20 µl mixture containing 4 µl of 5x reverse transcriptase buffer (250 mM TRI HCl pH 8.3 at 42°C), 50 mM MgCl₂, 250 mM KCl, 15 mM DTT, 10 U placental RNase inhibitor, 0.5 mM each dNTP, 50 pmol oligo-dT primer, and 20 U AMV (avian myeloblastosis virus) reverse transcriptase. After reverse transcription the HDC cDNA was amplified by polymerase chain reaction using two specific primers synthesized on a gene assembler plus (Pharmacia LKB): 5' primer, 5'-ATGATGG-AGCCCAGTGAATACC; 3' primer, 5'CCAGAATTCGCATGT-CTGAGG-TAG. Single-stranded cDNA mixture (4 µl) was supplemented with 50 pmol each of sense and antisense primers in a volume of 50 µl denatured for 2 min in a boiling bath and added to a 50 µl mix prewarmed at 72°C containing 0.25 mM each dNTP, 10 µl 10x Taq polymerase buffer (500 mM KCl, 100 mM TRIS-HCl pH 8.3 at 25°C, 15 mM MgCl₂ and 0.1% gelatin), and 1.5 U of Taq polymerase (Perkin-Elmer Cetus). The polymerase chain reaction program consisted of one cycle of 1 min at 94°C and 15 min at 72°C, followed by 35 cycles of 30 s at 94°C, 30 s at 55°C, and 4 min at 72°C, and was completed by an additional annealing at 55°C for 30 s and a final elongation at 72°C for 15 min. Polymerase chain reaction was performed in a Techne PHC-2 programmable heating block. Amplified products were purified by a glass-milk procedure (Geneclean BIO 101), blunt-ended with the Klenow fragment, and cut by EcoRI. The resulting blunt-end-EcoRI fragments were cloned in the p-MAL vector (Biolabs) cut by both StuI and EcoRI restriction enzymes. The resulting recombinant construct was transformed in the TB1 E. coli strain. Plasmid DNA was sequenced according to the manufacturer's protocol on the double-stranded DNA sequencing using sequenase V2.0. Plasmid containing amplified HDC cDNA was prepared according to the alkaline lysis method and purified on a CL4B column.

Northern blot analysis
Northern blot analysis was performed on enucleated tissue from the intramuscular injection sites of mice tested with hrRANTES (20 ng/50 µg), the negative (PBS) and positive controls (LPS), and anti-RANTES-Ab (50 µg/mouse) (R&D Systems). Actinomycin D was used on some tissue samples from RANTES i.m. injection sites.

Total RNA was isolated by guanidine hydrochloride as previously described (11, 28). Total RNA (10 µg/lane) was fractionated by electrophoresis on a formaldehyde denaturing agarose gel, transferred to nylon membranes (Hybond N, Amersham), and ³²P labeled HDC cDNA probe were hybridized (2x10⁸ cpm/mg). It was then washed four times at room temperature for 15 min in 2x solution of sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS), heated to 48°C for 30 min, and then washed twice in 0.1 xSSC and 0.1% SDS. Membranes were finally exposed to Kodak XAR5 for 3 days at -70°C. Signals were compared with ribosomal RNA to evaluate an equal quantity of RNA for each lane. A densitometric analysis was performed using a computerized image analyzer (Quantimed software, Leica, Heidelberg, Germany) for normalization of the relative mRNA levels (as reported in Results).

Histamine measurement
Histamine in tissue supernatants was estimated by a modification of the method described by Kaplan et al. (29). Tissues were minced in cold PBS, resuspended in water (1 g/0.2 ml), sonicated in a Branson 1200 ultrasound device for 10 min, vortexed for 5 min, and pelleted by 10 min centrifugation at top speed in an Eppendorf microcentrifuge. Total histamine in tissue homogenates taken from the injection sites was determined after centrifugation at 400 x g for 5 min. To 20 µl of supernatant taken from disrupted tissue was added 2 µl 20% perchloric acid, 6 µl 1 N NaOH, and 100 µl 0.05 M sodium phosphate, pH 7.4. The precipitate was pelleted by 5 min centrifugation at top speed in an Eppendorf microfuge and aliquots were taken from the supernatant for further histamine assay. In 50 µl of reaction mixture for histamine assay, 5–25 µl sample, 5 µl rat kidney histamine methyl transferase and S-(methyl-³H) adenosyl-L methionine (73.8 Ci/mM, Amersham) were present.

The isotope and enzyme were diluted with 0.05 M sodium phosphate pH 7.4 for optimal conditions. The reaction mixture was incubated 1 h at 37°C, after which 20 µl 1.5 M perchloric acid, 20 µl 10 M NaOH, and 500 µl freshly prepared toluene-isoamylalcohol (4:1 vol/vol) were added and the mixture shaken for 10 min. After 10 min centrifugation at 200 x g, 0.3 ml was taken from the upper phase and 4 ml of Aquasol (New England Nuclear, Boston, Mass.) scintillation fluid was added. Radioactivity was measured in a beta counter and the concentration of histamine was computed by using a standard curve. Samples were always performed in duplicate from two assay incubations. Histamine methyltransferase was a gift from the laboratory of Dr. David Cochrane (Medford, Mass.) and dinitrophenyl-bovine serum albumin was provided by Dr. Fu-Tong Liu, Scripps Clinic, La Jolla, Calif.

hrRANTES injection into the sole of the rat paw
Sprague-Dawley rats (Tacomic, Germantown, N.Y.) were used in all experiments. Before subcutaneous injection of RANTES or vehicle (PBS), the rats were fasted 24 h with free access to water. The experimental animals used were male, homogeneous for weight and age. Rats were anesthetized and injected subcutaneously with hrRANTES (20 ng/20 µl) or PBS (20 ng/20 µl) in the sole of the paw. After 4 h from injections, the animals were killed by inhalation of CO₂ and decapitated. The injected areas were uncovered and photographed; tissue was excised and slides were prepared with sections of the tissue. They were then stained with Toluidine blue (0.1% in 1% sodium borate for 2 min), and examined under an optical microscope (x20, x40, or x100) (Nikon Diaphore THD microscope, Japan).

Statistical analyses
Data from different experiments were combined and reported as the mean ±SD. Student's t test for independent means was used to provide statistical analyses (P>0.05 was considered as not significant).

	RESULTS

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Recruitment of mast cells after intraquadriceps injection of hrRANTES in vivo
hrRANTES, a monocytic, eosinophilic, and T lymphocytic chemotactic chemokine, is considered a potent inflammatory factor capable of activating basophils and bone marrow-derived murine mast cells (30). Intraquadriceps injection of hrRANTES 20 ng/50 µl produced a marked inflammatory response ( Fig. 1C) with mast cell accumulation 4 h after the injection, stained with mast cell-specific dye Toluidine blue. PBS (50 µl) (a negative control) ( Fig. 1A) was not effective in promoting inflammation, whereas LPS at 20 ng/50 µl (positive control) ( Fig. 1B) produced a strong inflammatory reaction. The cells were observed at a magnification of x20 under an optical microscope. When a goat IgG neutralizing antibody 50 µg/mouse (i.v.) was used 30 min before the injection of hrRANTES, the accumulation of mast cells was inhibited after 4 h ( Fig. 1D). Intramuscular injection site of RANTES 20 ng/50 µl in genetically mast cell-deficient (but not deficient in basophils) W/W^v mice did not provoke mast cell recruitment and inflammation ( Fig. 1E).

View larger version (636K): [in this window] [in a new window]   Figure 1. This is a representative photomicrograph (x20) of a quadriceps muscle tissue treated with A) PBS (50 µl (control); B) LPS 20 ng/50 µl (positive control); C) hrRANTES 20 ng/50 µl; D) anti-RANTES 50 µg/mouse (injected in the tail vein 30 min before hrRANTES was injected into the muscle); and E) hrRANTES injected in the muscle of genetically mast cell-deficient W/Wv mouse. Mast cells are darkly colored, stained with 0.1% Toluidine blue in biopsies of intraquadricept muscle tissue sites.

Ultrastructural studies
To better analyze mast cell accumulation in the muscle tissue exposed to RANTES, an electron microscopic study was performed using a PHILIPS 300 transmission electron microscope ( Fig. 2). The transmission electron micrograph (x24,300) is representative of four experiments, depicting mast cells from quadriceps muscle tissue after 4 h from hrRANTES injection. This figure shows three mast cells clumped together, which would be unusual in normal muscle tissue.

View larger version (149K): [in this window] [in a new window]   Figure 2. An electron microscopy study of mast cells from a representative experiment. Biopsied muscle tissue of injection site treated with 20 ng/50 µl hrRANTES after 4 h (the transmission electron micrograph is magnified x24,300).

Histamine production in muscle tissue injection sites in normal and genetically mast cell-deficient W/W^v mice and number of mast cells (normal mice) chemoattracted by RANTES
The increasing number of mast cells in the inflamed tissue leads to the augmentation of histamine levels in situ. RANTES has been presented as a potent chemotactic inflammatory cytokine. To better understand this effect, we studied the amount of histamine from sonicated tissue (1 g/0.2 ml) in the injected site of muscle tissue exposed to PBS (control), LPS (positive control), hrRANTES, IgG anti-RANTES, and RANTES injected in genetically deficient W/W^v mice ( Fig. 3A). The maximum histamine content of 25.0 ± 6 ng/ml and 15.0 ± 4.1 ng/ml was obtained when LPS 20 ng/50 µl or RANTES 20 ng/50 µl, was injected, whereas anti-RANTES treatment blocked the effect of RANTES and the levels of histamine from PBS treatment were very low (2.0 ± 1 ng/ml). In W/W^v mice, histamine was not detectable. The Student's t test for the effects of LPS (positive control) and RANTES was P < 0.01.

View larger version (23K): [in this window] [in a new window]   Figure 3. A) Total histamine level ± SD after intramuscular injection of 50 µl of PBS (control), LPS 20 ng (positive control), RANTES 20 ng, and anti-RANTES 50 µg/mouse (in 50 µl PBS) injected in the tail vein 30 min before hrRANTES treatments. B) The effect of intraquadricept injection of hrRANTES on chemotactic response of mast cells in normal and genetically mast cell-deficient W/Wv mice. This figure depicts the mean ± SD of 12 experiments where numerous mast cells are depicted in 200 mm2. Sections of the muscle injection site were stained with 0.1% Toluidine blue and analyzed under an optical microscope (x20). Mice were treated with PBS 50 µl (control), RANTES (20 ng/50 µl), anti-RANTES Ab (50 µl/mouse), and RANTES (20 ng/50 µl) injected into genetically mast cell-deficient W/Wv mice (last column). Mast cells were counted in an optic field using a grating size of 5 x 5 mm. P values (Student's t test) were obtained comparing control (*) with treatments. N.D., not detectable; N.S., not significant.

Figure 3B shows a histogram of mast cell counts (number of cells in 200 mm² at a magnification of x20) from muscle tissue injection sites. The infiltrated cells were stained with Toluidine blue (0.1%) and counted using a grated optic field. The animals were treated with PBS 50 µl (control, RANTES (20 ng/50 µl), LPS (20 ng/50 µl) as positive control, and IgG anti-hrRANTES (50 µg/mouse); cell counts were made for each of the following treatments: the last column represents results from hrRANTES-injected W/W^v mice. The highest chemoattractant effect (87 ± 10) was obtained when 20 ng/50 µl LPS was injected, whereas hrRANTES produced 52 ± 10 mast cells. The anti-RANTES treatment (30 min) before hrRANTES injection inhibited the effect of hrRANTES; in W/W^v mice treated with hrRANTES, the number of cells was not detectable. Student's t test were calculated comparing control with treated animals.

Increase of HDC mRNA levels by RANTES
Since it was previously shown that RANTES is chemoattractive for mast cells (30) and HDC is an important marker for the production of histamine, a probe was prepared to detect mRNA encoding the HDC gene. Oligonucleotide primers specific for HDC sequences were used to amplify a product from rat brain cDNA, and this yielded a 1019 bp fragment of DNA after digestion with EcoRI restriction enzyme. After cloning into the p-Mal plasmid, this was used to detect HDC mRNA by Northern blot hybridization (14).

Figure 4 shows the effect of 20 ng/50 µl hrRANTES, antiRANTES (50 µg/mouse), LPS 20 ng (positive control), and PBS 50 µl (negative control) on histidine decarboxylase mRNA expression in quadriceps muscle injection sites of mice. Lane 1 represents histidine decarboxylase mRNA of biopsied muscle injection site after 4 h treatment with PBS (50 µl). Lane 2 represents LPS treatment (positive control); lane 3 shows the response of RANTES at 20 ng/50 µl, lane 4 represents injection of anti-RANTES antibody 30 min injected before RANTES; lane 5 represents RANTES injected in W/W^v mice. Four hours after the injection, steady-state levels of HDC mRNA in PBS treatment were low ( Fig. 4, lane 1), whereas in the RANTES-treated tissue there was a marked effect on HDC mRNA compared to the controls. Expressed levels of HDC mRNA were high in the LPS treated tissues. IgG anti-RANTES antibody treatment inhibited the effect of RANTES, whereas in W/W^v mice the effect of RANTES was not detectable. When actinomycin D was used (10^-6 M) it significantly inhibited expression of mRNA transcripts, suggesting that RANTES appears to control HDC gene expression at a transcriptional level.

View larger version (45K): [in this window] [in a new window]   Figure 4. Effect of intramuscular injections of PBS 50 µl (lane 1), LPS 100 ng/50 µl (lane 2), RANTES 20 ng/50 µl (lane 3), anti-RANTES 50 µg/animal (lane 4), RANTES 20 ng/50 µl (lane 5) in genetically mast cell-deficient W/Wv mice, and actinomycin D 10-6 M (lane 6) on histidine decarboxylase (HDC) mRNA expression after 4 h of treatment.

Subcutaneous injection of hrRANTES into the sole of the rat paw
In previous studies (7, 8) we reported that intradermal injections of hrRANTES produced a strong inflammatory reaction after 4 h. Subcutaneous injection of hrRANTES (20 ng/20 µl) into the sole of the rat paw caused a prominent inflammatory reaction compared to the control, PBS (20 µl) -treated paw. Mast cell recruitment was confirmed in biopsy tissue stained with Toluidine blue, where a high number of mast cells were shown compared with controls (PBS; Fig. 5).

View larger version (278K): [in this window] [in a new window]   Figure 5. A) Microscopic observation of mast cell migration from tissue taken from the rat sole paw induced by hrRANTES 20 ng/20 µl stained with Toluidine blue and photographed (x40). B) Control (PBS 20 µl) with some resident mast cells (darkly colored spots).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mast cells (unique for their metachromasia) and basophils are two principal cell types involved in the initiation of human allergic reactions by virtue of possessing high-affinity receptors for IgE in their plasma membranes (31, 32). Previously, it was reported that cytokines, such as interleukin 3 or protein c -kit receptor ligand, have the capacity to induce mast cell motility (33, 34). Here we demonstrated that intraquadriceps injection of hrRANTES in mice causes mast cell recruitment, as evidenced by staining with Toluidine blue dye. This effect was specifically inhibited by an anti-RANTES antibody injected intravenously 30 min before the intramuscular injection of hrRANTES.

When genetically mast cell-deficient W/W^v mice were used, the intraquadriceps injection of human RANTES did not provoke any appreciable inflammatory reaction or basophilic cell extravasation. Our results clearly demonstrate that hrRANTES acts mainly on mast cell recruitment in producing inflammation, which are bone marrow-derived tissue mast cells (30). Moreover, we found that intramuscular injections of hrRANTES in normal mice induce histamine generation, due to the accumulation of mast cells recruited by hrRANTES, which was abolished when an anti-RANTES antibody was used. This effect was similar to the injection of LPS when compared to the controls. Histamine levels most likely reflect mast cell numbers recruited by RANTES from resident, bone marrow-derived tissue mast cells (30). Other inflammatory cell types recruited by RANTES, such as eosinophils, may have contributed to the histamine levels observed in these studies. These cells have been characterized in our previous study (7). The level of histamine in biopsied tissue from genetically mast cell-deficient W/W^v mice was undetectable.

In addition, we found that RANTES induces HDC expression, which was evidenced at the mRNA level compared with the controls. This effect was abolished in the presence of actinomycin D, which inhibits mRNA transcription. HDC mRNA is presumably generated locally in the affected tissue by both resident (control) and immigrant mast cells (experimental). Again, this effect was inhibited by an anti-RANTES antibody in normal mice and was absent in genetically mast cell-deficient W/W^v mice. Here the accumulation of mast cells was maximal at 4 h after RANTES or LPS injections, whereas after 16 or 24 h there was no difference (data not shown). The inflammatory potential and recruitment of mast cells by hrRANTES was confirmed when injected into the sole of a rat paw model. Other authors (11, 20) also found maximal white cell infiltration at 4 h after chemokine challenge into the site of injection (11, 28). In another study, Taub et al. (20) found that mast cells and basophils migrate in response to chemokine stimuli, suggesting that some chemokine receptor function are shared among these cell populations. In our study, only mast cells in normal mice were recruited by hrRANTES, whereas circulatory basophils from genetically mast cell-deficient W/W^v mice were not chemoattracted in the RANTES injection sites, suggesting the specificity of this chemokine for mast cells. We believe that our results might be only the tip of a large iceberg since many authors have reported the lack of specificity of many diverse chemokines, particularly in white cell chemoattraction. Our observations suggest that RANTES represents an important mediator of tissue mast cell recruitment in the setting of inflammatory reactions. The ability of RANTES to stimulate mast cell chemoattraction and histidine decarboxylase mRNA argues for a role of RANTES in mast cell function and other aspects of mast cell response.

Our studies contribute to an understanding of the mechanisms by which mast cells profoundly effect acute inflammatory responses in vivo and suggest that the antagonist(s) of RANTES may have inhibitory biological effects on inflammatory conditions. Moreover, these results provide direct in vivo evidence that hrRANTES has significant proinflammatory activity on mast cells in normal mice, whereas this effect was not detectable in genetically mast cell-deficient W/W^v mice with mutant alleles at the W locus. In addition, RANTES may be an important mediator in atopic pathologies characterized by specific activation of mast cells. However, more studies of the effect of RANTES in vivo and in vitro are required to clarify the specificity of the proinflammatory effect of this and other chemokines. To better understand the new evidence revealed by the data reported here, studies involving the antagonism of RANTES through competitive receptor binding are under way.

	ACKNOWLEDGMENTS

 
This study was supported by a grant from the Ministry of University, Scientific and Technological Research, Italy, 1996 (40 and 60%), and in part by a grant from Muro Pharmaceutical (Tewksburg, Mass.). We wish to thank Dr. Alfredo Grilli, Biology Department, University of Chieti, for realization of the photos provided for this manuscript, and Mr. William Boucher for technical assistance.

	FOOTNOTES

 
¹ Correspondence: Immunology Division, University of Chieti School of Medicine, Via dei Vestini, 66013 Chieti, Italy. E-mail: pconti{at}unich.it

² Abbreviations: HDC, histidine decarboxylase; hr, human recombinant; IgE, immunoglobulin E; LPS, lipopolysaccharide; MCP-1, monocyte chemotactic protein 1; PBS, phosphate-buffered saline; RANTES, regulated upon activation, normal T cell expressed and presumably secreted; SDS, sodium dodecyl sulfate; SSC, solution of sodium citrate.

Received for publication April 28, 1998. Revision received July 15, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Oppenheim, J. J ., Zacharie, C. O. C., Mukaida, N., and Matsushima, K. (1991) Properties of the novel proinflammatory supergene `Intercrine' cytokine family. Annu. Rev. Immunol. 9, 617–623[Medline]
2. Baggiolini, M., Dewald, B., and Moser, B. (1994) Interleukin-8 and related chemotactic cytokines—CXC and CC chemokines. Adv. Immunol. 55, 97–103[Medline]
3. Baggiolini, M., and Dahinden, C. A. (1994) CC chemokines in allergic inflammation. Immunol. Today 15, 127–132[Medline]
4. Armsted V. E., Minchenko, A. G., Campbell, B., and Lefer, A. M. (1997) P-selectin is up-regulated in vital organs during murine traumatic shock. FASEB J. 11, 1271–1279[Abstract]
5. Schall, T. T., Bacon, K., Karen, J. T., and Goeddel, D. V. (1990) Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature (London) 347, 669–673[Medline]
6. Schall, T. T. (1991) Biology of the RANTES/SIS cytokine family. Cytokine 3, 165–172[Medline]
7. Conti, P., Pang, X., Boucher, W., Letourneau, R., Reale, M., Barbacane, R. C., Thibault, J., and Theoharides, T. C. (1997) Impact of RANTES and MCP-1 chemokines on in vivo basophilic cell recruitment in rat skin injection model and their role in modifying the protein and mRNA levels for histidine decarboxylase. Blood 89, 4120–4127[Abstract/Free Full Text]
8. Conti, P., Reale, M., Barbacane, R. C., Frydas, S., Felaco, M., Grilli, A., Placido, F. C., Cataldo, I., Feliciani, C., Di Gioacchino, M., Anogianakis, G., Dimitriadou, D., Vacalis, D., and Trakatellis, A. (1997) Massive infiltration of basophilic cells in inflamed tissue after injection of RANTES. Immunol. Lett. 58, 101–106[Medline]
9. Conti, P., Pang, X., Boucher, W., Letourneau, R., Reale, M., Barbacane, R. C., Thibault, J., and Theoharides, T. C. (1997) Rantes is a proinflammatory chemokine and chemoattracts basophilic cells to extravascular sites. J. Pathol. 183, 352–358[Medline]
10. Ying, S., Toborda-Barata, L., Meng, Q., Humbert, M., and Bay, A. B. (1995) The kinetics of allergen-induced transcription of messenger RNA for monocyte chemotactic protein-3 and RANTES in the skin of human atopic subjects: relationship to eosinophil, T cell, and macrophage recruitment. J. Exp. Med. 181, 2153–2163[Abstract/Free Full Text]
11. Meurer, R., Van Riper, G., Feeney, W., Cunningham, P., Hora, D., Jr., Springer, M. S., Euan MacIntyre, D., and Rosen, H. (1993) Formation of eosinophilic and monocytic intradermal inflammatory sites in the dog by injection of human RANTES but not human monocyte chemoattractant protein 1, human macrophage inflammatory protein 1a, or human interleukin 8. J. Exp. Med. 178, 1913–1920[Abstract/Free Full Text]
12. Theoharides, T. C., Bondy, P. K., Tsakalos, N. D., and Askanase, P. W. (1982) Differential release of serotonin and histamine from mast cells. Nature (London) 297, 229–235[Medline]
13. Theoharides, T. C., Seighart, W., Greengard, P., and Douglas, W. W. (1980) Antiallergic drug cromolyn may inhibit histamine secretion by regulating phosphorylation of a mast cell protein. Science 207, 80–83[Abstract/Free Full Text]
14. Conti, P., Boucher, W., Letourneau, R. , Feliciani, C., Reale, M., Barbacane, R. C., Vlagopoulos, P., Bruneau, G., Thibault, J., and Theoharides, T. C. (1995) Monocyte chemotactic protein 1 (MCP-1) is a potent histamine and [³H]-5HT-releasing factor for mast cells and provokes cell aggregation. Immunology 86, 434–440[Medline]
15. Frank Austen, K. (1982) Tissue mast cells in immediate hypersensitivity. Hosp. Pract. 17, 98–108
16. Conti, P., Boucher, W., Feliciani, C., Mammarella, S., Kudchadker, L., Barbacane, R. C., Reale, M., Haggag, I., Bruneau, G., Thibault, J., and Theoharides, T. C. (1995) Effect of interleukin-1 receptor antagonist (IL-1RA) on histamine and serotonin release by rat basophilic leukemia cells (RBL-2H3) and peritoneal mast cells. Mol. Cell. Biochem. 155, 61–68
17. Conti, P., Reale, M., Barbacane, R. C., Panara, M. R., Bongrazio, M., and Theoharides, T. C. (1992) Role of lipoxins A4 and B4 in the generation of arachidonic acid metabolites by rat mast cells and their effect on [³H]serotonin release. Immunol. Lett. 32, 117–124[Medline]
18. Kuna, P., Reddigari, S. R., Schall, T. J., Ricinski, D., Viksman, M. Y., and Kaplan, A. P. (1992) RANTES a monocyte and T lymphocyte chemotactic cytokine releases histamine from human basophils. J. Immunol. 149, 636–642[Abstract]
19. Bischoff, S. C., Krieger, M., Brunner, T., Rot, A., Tscharner, V., Baggiolini, M., and Dahinden, C. A. (1993) RANTES and related chemokines activate human basophil granulocytes through different G protein-coupled receptors. Eur. J. Immunol. 23, 761–766[Medline]
20. Taub, D., Proost, P., Murphy, W. J., Anver, M., Longo, D. L., Van Damme, J., and Oppenheim, J. J. (1995) Monocyte chemotactic protein-1 (MCP-1) -2 and -3 are chemotactic for human T lymphocytes. J. Clin. Invest. 95, 1370–1376
21. Uguccioni, M., D'Apuzzo, M., Loetscher, M., Dewald, B., and Baggiolini, M. (1995) Action of the chemotactic cytokine MCP-1, MCP-2, MCP-3, RANTES, MIP-1 and MIP-1ß on human monocytes. Eur. J. Immunol. 25, 64–68[Medline]
22. Kitamura, Y., Go, S., and Hatanaka, K. (1978) Decrease of mast cells in W/W^v mice and their increase by bone marrow transplantation. Blood 52, 447–452[Abstract/Free Full Text]
23. Nakano, T., Kanakura, Y., Nakahata, T., Matsuda, H., and Kitamura, Y. (1987) Genetically mast cell-deficient W/W^v mice as a tool for studies of differentiation and function of mast cells. Federation Proc. 46, 1920–1923[Medline]
24. Galli, S. J., and Kitamura, Y. (1987) Genetically mast cell-deficient W/W^v and Sl/Sl^d mice. Their value for the analysis of the roles of mast cells in biologic responses in vivo. Am. J. Pathol. 127, 191–198[Medline]
25. Go, S., Kitamura, Y., and Nishimura, M. (1980) Effect of W and W^v alleles on production of tissue mast cells in mice. J. Heredity 71, 41–44[Free Full Text]
26. Dimitriadow, V., Buzzi, M. G., Moskowitz, M. A., and Theoharides, T. C. (1991) Trigeminal sensory fiber stimulation induces morphologic changes reflecting secretion in rat dura mast cells. Neuroscience 44, 97–102[Medline]
27. Bruneau, G., Nguyen, V.-C., Gros, F., Bernheim, A., and Thibault, J. 1992. Preparation of a rat brain histidine decarboxylase (HDC) cDNA probe by PCR and assignment of the human HDC gene to chromosome 15. Human Genet. 90, 235–240
28. Choczynski, P., and Sacchi, N. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–162
29. Kaplan, A. P., Haak-Frendscho, M., Fauci, A., Dinarello, C. A., and Malbert, E. (1985) A histamine releasing factor from activated human mononuclear cells. J. Immunol. 135, 2027–2032[Abstract]
30. Taub, D., Dastych, J., Inamura, N., Upton, J., Kelvin, D., Metcalf, D., and Oppenheim, J. J. (1995) Bone marrow derived murine mast cells migrate, but do not degranulate in response to chemokines. J. Immunol. 154, 2393–2402[Abstract]
31. Kay, A. B., Ying, S., Varney, V., Gaga, M., Durham, S .R., Moqbel, R., Wardlaw, A. J., and Hamid, Q. (1991) Messenger RNA expression of the cytokine gene cluster, interleukin-3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects. J. Exp. Med. 173, 775–778[Abstract/Free Full Text]
32. VanOtteren, G. M., Streiter, R. M., Kunkel, S. L., Paine, R., III, Greenberger, M. J., Danforth, J. M., Burdick, M. D., and Standiford, T. J. (1995) Compartmentalized expression of RANTES in a murine model of endotoxemia. J. Immunol. 154, 1900–1908[Abstract]
33. Matsura, N., and Zetter, B. R. (1989) Stimulation of mast cell chemotaxis by interleukin 3. J. Exp. Med. 170, 1421–1427[Abstract/Free Full Text]
34. Meininger, C. J., Yao, H., Rottapel, R. , Bernstein, A., Zsebo, K. M., Zetter, B. R. (1992) The c-kit receptor ligand as a mast cell chemoattractant. Blood 79, 958–963[Abstract/Free Full Text]

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