Department of Anesthesia, Graduate School of medicine The University of Tokyo, Tokyo, Japan.

--------------------------------------------------------------------------------
Abstract

We report here canine mastocytoma-derived cells (CMMC) activation via two pentraxin, limulus and human CRP. Mast cell chemotaxis was measured by the Boydenﾕs blindwell chamber. To confirm the cell migration was chemotactic, ﾒcheckerboardﾓ analysis was performed. We used Fura-2 to investigate CRP-mediated cytosolic calcium elevation. To examine whether CRP-induced stimulation is mediated through G-proteins, CMMC were incubated with pertussis toxin (PTx) before use in chemotaxis assay and Ca2+ mobilization. CMMC migration in response to CRP was both chemokinetic and chemotactic. Limulus-CRP induced a transient Ca2+-mobilization dose-dependently. Preincubation of the cells with PTx inhibited CRP chemotaxis and Ca2+-mobilization, suggesting that G-proteins of the Gi-class are involved in the chemotaxis. We suggest that CRP may participate in the migration of mast cells to inflamed tissues during an acute-phase response. CRP-mediated recruitment of mast cells might play an important role in hypersensitivity and inflammatory processes.

Introduction

Mast cell accumulation occurs in certain inflammatory conditions that include host response to microbes, perioperation tissue repair and fibrosis, delayed hypersensitivity reactions, neoplasia, allergy, and in chronic conditions such as inflammatory bowel disease, rheumatoid arthritis, asthma [1] .Mature mast cells are resident cells found in most tissues. For long time, they have been considered as stationary cells with no property to migrate. [1.2]. However, the factors that control migration of mast cells to sites of inflammation and tissue repair remain largely undefined. Directed migration (chemotaxis) of mast cells within tissue may be the most speedy and reasonable mechanism, which increase the number of mast cell. The use of chemotaxis assay has allowed the identification of several mast cell chemoattractants. In murine mast cells, such as laminin [3], interleukin-3 (IL-3)[4], stem cell factor (SCF)[5], transforming growth factor_(TGF_)[6], and monocyte chemotactic protein-1 (MCP-1)[7] were found to promote mast cell migration. In contrast, SCF [8], anaphylatoxins (C3a and C5a) [9] have been reported to induce chemotaxis in human mast cells.
Some of the pathological conditions that induce mast cell hyperplasia are also associated with high serum concentrations of acute phase protein such as CRP. Serum concentrations of CRP in humans increase from less than one to hundreds of mg/ml as part of the inflammatory response to perioperation period, infection or injury [10]. It has been said that CRP was synthesed only in the liver, but CRP is expressed in macrophages [11]. Recent reports have indicated that CRP is not just a marker of underlying inflammation, but also is directly involved in the pathogenesis of arteriosclerotic lesions [12.13], the tissue damage that accompanies acute myocardial infarction [14] and the trigger of attack of intrinsic infectious type asthma.
We have already demonstrated that IgG-mediated histamine release from canine cutaneous mastocytoma-derived cells (CM-MC) [15] and have investigated the expression of Fc_ receptors on the cell, monomeric IgG-binding to the cell and IgG-mediated or IgE-mediated signal transduction in CM-MC [16]. In the present study, we used a modified Boydenﾕs blindwell chamber chemotaxis assay to evaluate the potency of CRP as a chemoattractant for CMMC. We propose CRP as an additional group of chemotactic factors through which CMMC is recruited to sites of inflammation.

Materials and Methods

Cells and buffers
The CMMC derived from canine cutaneous mastocytoma were passaged in the RPMI 1640 medium with 10% heat-inactivated FCS. The Pipes-ACM buffer used for the experiments consisted of 140 mM NaCl, 5mM KCl, 0.6mM MgCl2, 1mM CaCl2, 5.5mM glucose, 0.1% BSA and 10mM Pipes (pH 7.4). Human-CRP were from (Chemicon, Temecula, Canada); Limulus-CRP from (Sigma, St.Louis, USA). All concentration of CRP was nontoxic for CMMC assessed by trypan blue.
CMMC chemotaxis checkerboard analysis
CMMC chemotaxis was measured by the Boydenﾕs blindwell chamber technique using a 48-well multi-well chamber, and an 8-_m-pore-size membrane (pvp free) (Neuroprobe, Gaithersburg, MD). Cell migration was expressed as the average of five randomly selected high power fields per well. To confirm that the cell migration was chemotactic, ﾒcheckerboardﾓ analysis was performed. Each combination was tested in triplicate. The location of the various combinations in the 48-well manifold was randomized. The length of time required for CMMC chemotaxis to CRP was measured by varying incubation time of the chemotaxis chambers. A total of 100_g/ml of CRP was used to examine whether CRP-induced stimulation is enhanced by IgG cross linking. CMMC were activated ,in some experiments, by the additions of goat anti-canine IgG (10_g/ml, Bethyl, Montgomery, TX, USA) in upper wells after sensitized with canine IgG (10_g/ml, ICN Pharmaceuticals Inc., Aurora, OH,USA) for 30 minutes at 37｡C. The chemotaxis inhibition was measured by using various concentrations (10_g/ml to 200_g/ml) of goat anti-human CRP (Bethyl, Montgomery, TX, USA) before human CRP stimulation. A various concentrations of goat anti-human CRP was added to 100_g/ml human CRP for 30 minutes at 37｡C before chemotaxis. CMMC were incubated with 10,30,100 ng/mL of PTx (Sigma Chemical Co, St Louis, MO) in FCS complete medium for 120 minutes at 37｡C. The cells were then washed twice and resuspended in complete medium before use in chemotaxis assay.
Measurement of the intracellular calcium mobilization and histamine release
[Ca2+]i was calculated as described in a previous paper［17］. Ca2+ mobilization preincubation with 10, 30,100 ng/mL of PTx were also measured. Histamine were measured using o-phthalaldehyde fluorometric technique modified for autoanalysis as described ［18］.

Results and discussion

Chemotaxis response of CMMC to limulus and human CRP
The chemotaxic activity of CRP for canine mast cell was determined in a modified Boydenﾕs chamber. As shown in Table 1 (limulus) and 2 (human), chemokinesis was present, since even when no gradient existed, the numbers of cells that migrated increased as the concentration of CRP increased. The number of migrated cells per hpf was 13.3 ｱ 0.6 (with limulus CRP) ,11.0ｱ1.0 (with human CRP) and 0.7 ｱ 0.6 (without stimulation),respectively. Cell migration was measured in various concentration of CRP, both in the presence and absence of gradients of chemoattractant. The cell migration was chemotactic, since migrated cell increased in the presence of gradient. Thus, CMMC migration in response to CRP was both chemokinetic and chemotactic. To examine the effect of extracelluler Ca2+ concentration, we used Ca2+ free solution (Pipes EGTA) and Pipes ACM (Ca2+=3mM) instead of Pipes ACM (Ca2+=1mM).The number of migrated cells per h.p.f. was no difference (data not shown). Chemotaxis to CRP was independent on extracelluler Ca2+ concentration.
Figure 1a (limulus) and 1b (human) show a typical bell-shaped dose response curve of mast cell migration with higher concentrations resulting in the loss of directed migration. However, in contrast to other mast cell chemoattractants such as SCF and C3a [8.9], CRP induced mast cell migration in a more narrow concentration range. The optimal concentration of CRP for the induction of mast cell migration was 250_g/ml, which is higher than normal serum level (<1_g/ml), but well below the levels seen in inflammatory conditions. Figure 1c (limulus) and 1d (human) show the number of CMMC increased with time and reached plateau at 2 h. As mast cell chemotaxis to CRP is enhanced by mast cell activation, we also determined if activation by IgG-dependant stimuli would enhance their chemotactic responsiveness. Figure 1e (limulus) and 1f (human) show CMMC chemotaxis to CRP is mildly enhanced after activation of CMMC with anti-IgG after sensitization of the cells with IgG.
To examine whether CRP-induced stimulation is mediated through guanine nucleotide-binding proteins (G proteins), CMMC were incubated with pertussis toxin before use in the chemotaxis assay. Pretreatment with PTx (10ng/ml) partially inhibited the migratory response to limulus-CRP (100_g/ml) and PTx (100ng/ml) totally inhibited (Figure 2a). Thus, chemotactic response of mast cells to CRP is pertussis toxin sensitive. As shown in Figure 2b, pretreatment with goat anti-human CRP (50_g/ml to 200_g/ml) partially abrogated the migratory response to 100_g/ml of limulus-CRP in a dose-dependent manner.
Measurement of the intracellular calcium mobilization and histamine release
In various cell types, migration was found to be associated with alteration in [Ca2+] i, as an early step in signal transduction [19]. Therefore, [Ca2+] i changes in CMMC in response to CRP were measured. CRP-mediated intracellular calcium mobilization was examined using the calcium indicator Fura-2-AM as shown in (Figure 3 a.b). The [Ca2+] i increase by CRP stimulation occurred and the rapid increase in the early interval merged into a very slow increment. Limulus-CRP (100 and 300_g/ml) induced a rapid and transient Ca2+ mobilization dose-dependently. In contrast, no significant elevation of [Ca2+]i was observed in resting cells (data not shown). Ca2+ mobilization of CMMC in response to CRP was differ from anti-IgG activation (Figure 3c). To examine whether CRP-induced stimulation is mediated through guanine nucleotide-binding proteins (G proteins), CMMC cells were incubated with pertussis toxin before use in measurement of the intracellular calcium mobilization. The [Ca2+] i elevation response to limulus-CRP was partially abolished pretreatment with PTx (10ng/ml) and totally abolished by PTx (100ng/ml) (Figure 4 a b c d).
Limulus and human CRP induced histamine release. The maximum histamine release from CMMC was 5.1ｱ0.9% at 300_g/ml of limulus-CRP and 6.9ｱ0.5% at 300_g/ml of human-CRP (Figure.5). RBL-2H3 (rat basophilic leukemic cell-2H3) and human basophils did not release histamine by limulus and human CRP.
The main biologic function of CRP lies in its ability to recognize pathogens and damaged cells of the host and to mediate their elimination by recruiting the complement system and phagocytic cells. Function of CRP attributable to direct interactions with phagocytic cells have been known for many years [20], but it was only recently that CRP binds to the high affinity IgG receptor, Fc_RI, on mononuclear cells and on COS cells transfected with a cDNA encoding this receptor [21.22]. In our study, CM-MC cell chemotaxis was enhanced after activation of CMMC with anti-IgG after sensitization of the cells with IgG.(Figure 3c). In addition, pretreatment with PTx inhibited CRP-mediated chemotaxis of CMMC (Figure 4) and the Ca2+ mobilization, indicating that guanine nucleotide-binding proteins (Gi proteins) are involved in signal transduction pathways in CMMC. These results indicate that mast cell chemotaxis is mediated by distinct signal transducing pathways from immuno-receptor mediated one.
We used two of pentraxin family, limulus and human CRP. The cross-reactivity of limulus CRP with human one existed due to antigenic similarity between limulus and human CRP [23]. Limulus CRP shares many of the biological functions of immunoglobline such as complement fixation and phagocytes [24]. The cording regions of the limulus CRP and human CRP genes share approximately 30% identity and two stretches of highly conserved regions, one of which falls in the region proposed as the phosphorylcholine binding site, while the other site is very similar to the consensus sequence required for calcium binding in calmodulin and related protein. [24.25]. In conclusion, we propose CRP is one of the G protein-mediated chemoattractants which governs mast cell distribution and promotes their accumulation in acute inflammatory phase.

Figure legend

Table1 (limulus) Table 2 (human)
In the top wells of the chemotactic chamber, various concentrations of CRP (limulus and human) (0, 50,100, and 200_g/ml) were applied together with the target cells. In the bottom wells, the same concentrations of CRP were placed such that all possible combinations above and below the filter were tested.
Figure1
Limulus-CRP was placed into each of the bottom wells; above the filter CMMC prepared as described were placed into each of the top wells. The Chambers were then incubated at 37ﾟC, 5% CO2 for 2h. After incubation, cells on the top of the filter were removed by scraping. The filter was then stained with Diff Quick stain. CMMC chemotactic activity was determined as the total number of migrated CMMC counted in 10 hpf (high power field) average using a light microscope (BX51,Olympus,Osaka,Japan) at 400_ magnification.
a (limulus) b(human)
Dose-dependent chemotaxis of CMMC to CRP.
Vertical axis: CMMC chemotaxis expressed as number of cells migrated of per hpf. Horizontal axis: concentration of CRP. Bar represents SEM for three individual experiments.
c (limulus) d(human)
Time dependence chemotaxis of CMMC to CRP (100_g/ml)
CMMC demonstrated chemotactic migration in response to 100_g/ml of CRP. Vertical axis: CMMC chemotaxis expressed as number of cells migrated per hpf. Horizontal axis: incubation time of chemotaxis chamber. Bar represents SEM for three individual experiments.
e (limulus) f(human)
CMMC were activated by the additions of canine anti-IgG after sensitized with canine IgG
Migration of IgG activated CMMC cell to limulus CRP. Open circles represent the inclusion of anti-IgG (10_g/ml) after sensitized with IgG (10_g/ml). Bar represents SEM for three individual experiments.
Figure 2
a
The chemotaxis inhibition was measured by using various concentrations (50_g/ml to 200_g/ml) of goat anti-human CRP. before human CRP stimulation. A various concentrations of goat anti-human CRP was added to 100_g/ml human CRP for 30 minutes at 37｡C before chemotaxis. Pretreatment with goat anti-human CRP (50_g/ml to 200_g/ml) partially inhibited the migratory response to limulus-CRP (100_g/ml) decreased in a dose-dependent manner
b
CMMC were incubated with PTx before use in the chemotaxis assay. Limulus CRP stimulates migration of CMMC via a PTx-sensitive G protein. Pretreatment with PTx (10ng/ml) partially inhibited the migratory response to limulus-CRP (100_g/ml) (71% inhibition, P = 0.05) and PTx (100ng/ml) totally inhibited (100% inhibition).
Figure 3
CMMC (5 x 105 cells/ml) were resuspended in 1.5ml of the buffer and were stimulated with limulus-CRP (30,100 and 300_g/ml) for 30 min at 37 ﾟC. The cells were loaded with Fura-2-AM (6_M, Dojindo, Kumamoto, Japan) for the last 15 min of the stimulation. During the measurement of [Ca2+]i, cells were stirred at 37 _ and illuminated alternately with two excitation wavelengths (340 and 380 nm) and fluorescence at 495 nm was measured with a Shimazu RF-5000 spectrophotometer (Kyoto, Japan).
Stimulated with limulus CRP 100_g/ml (a) Stimulated with limulus CRP 300_g/ml (b) a rapid [Ca2+] i increase was observed in the CM-MC cells sensitized with 10_g/ml of canine IgG and with stimulation with 10_g/ml of anti-canine IgG. (Positive control) (c)
Figure 4
After treatment of CMMC with PTx at (0.1.10.100) ng/ml for 120 minutes, cells were stimulated with limulus-CRP (300_g/mL) Pretreatment with PTx (100ng/ml) totally inhibited (100% inhibition) the [Ca2+] i elevation response to limulus-CRP.
Figure 5
Histamine release from CMMC induced by limulus CRP (solid line) and Human CRP (broken line). Cells were incubated with the stimuli at 3ﾟC for 30 min. Bar represents SEM for three individual experiments.

References

1. Weber S, Kruger-Krasagakes S, Grabbe J , Zuberbier T, Czarnetzki BM, Mast cells, Int J Dermatol. 34(1995)1-10.
2. Ann M.Dvorak ,New aspects of mast cell biology, Int. Arch Allergy Immunol. 114(1997)1-9.
3. Thompson HL, Burbelo PD, Yamada Y, Kleinman HK, Metcalfe DD, Mast cells chemotax to laminin with enhancement after IgE-mediated activation. J. Immunol. 143 (1989) 4188-4192.
4. Matsuura N, Zetter BR, Stimulation of mast cell chemotaxis by interleukin 3, J. Exp. Med. 170 (1989) 1421-1426.
5. Meininger CJ, Yano H, Rottapel R , Bernstein A, Zsebo KM, Zetter BR, The c-kit receptor ligand functions as a mast cell chemoattractant, Blood 79 (1992) 958-963.
6. Gruber BL, Marchese MJ, Kew RR, Transforming growth factor- 1 mediates mast cell chemotaxis, J Immunol. 152 (1994) 5860-5867.
7. Taub D, Dastych J, Inamura N Upton J, Kelvin D, Metcalfe D, Oppenheim J, Bone marrow-derived murine mast cells migrate, but do not degranulate, in response to chemokines, J Immunol 154(1995) 2393-2402.
8. Nilsson G, Butterfield JH, Nilsson K , Siegbahn A, Stem cell factor is a chemotactic factor for human mast cells, J Immunol 153 (1994) 3717-3723.
9. Karin Hartmann, Beate M. Henz, Sabine Kruger-Krasagakes, J嗷g K喇l, Reinhard Burger, Sven Guhl, Ingo Haase, Undine Lippert, and Torsten Zuberbier, C3a and C5a Stimulate Chemotaxis of Human Mast Cells ,Blood 89 (1997) 2863-2870.
10. Gabay,C and I.Kushner, Acute-phase proteins and other systemic responses to inflammation, N. Engl. J. Med. 340 (1999) 448-454.
11. Dong and J.R. Wright, Expression of C-reactive protein by alveolar macrophages. J. Immunol, 156 (1996) 4815-4820.
12. Bernhard Metzler,Qingbo Xu, The role of mast cells in atherosclerosis, Int Arch Allrgy Immunol 114 (1997) 10-14.
13. Zwaka, V. Hombach and J. Torzewski, C-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis, Circulation 103 (2001) 1194・197.
14. Griselli, J. Herbert, W.L. Hutchinson, K.M. Taylor, M. Sohail, T. Krausz , M.B. Pepys , C-reactive protein and complement are important mediators of tissue damage in acute myocardial infarction, J. Exp. Med. 190 (1999) 1733・739.
15. Takahashi T, Kitani S, Nagase M, Mochizuki M, Nishimura R, Morita Y, Sasaki N, IgG-mediated histamine release from canine mastocytoma-derived cells, Int Arch Allergy Immunol 125 (2001) 228-235.
16. Sato Y, Teshima R, Nakamura R, Sasaki N, Morita Y, Sawada J, Kitani S, IgG-mediated signal transduction in canine mastcytoma-derived cells, Int Arch Allergy Immunol (in press)　
17. Teshima R, Ikeuchi H, Terao T, Nakanishi M, The effect of staurosporine on Ca2+ signals in rat basophilic leukemia (RBL-2H3) cells, Chem Pharm Bull 39 (1991) 747-751.
18. Kitani S, De Silva NR, Morita Y, Teshima R, Global environmental pollutant substance vanadium activates mast cells and basophils at the late phase in the presence of hydrogen peroxide, Environ. Toxicol.Pharmacol. 6 (1998) 1-12.
19. Benyon RC, Lowman MA, and Church MK, Human skin mast cells, Their dispersion, purification, and secretory characterization, J Immunol 138(1987) 861-866.
20. Mortensen, A.P. Osmand, T.F. Lint and H. Gewurz , Interaction of C-reactive protein with lymphocytes and monocytes: complement-dependent adherence and phagocytosis, J. Immunol. 117 (1976) 774・81.
21. Crowell, T.W. Du Clos, G. Montoya, E. Heaphy and C. Mold, C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc RI, J. Immunol. 147 (1991) 3445・451.
22. Marnell, C. Mold, M.A. Volzer, R.W. Burlingame T.W. Du Clos , C-reactive protein binds to Fc RI in transfected COS cells, J. Immunol. 155 (1995) 2185・193.
23. Ying SC, Marchalonis JJ, Gewurz AT, Siegel JN, Jiang H, Gewurz BE, Gewurz H, Reactivity of anti-human C-reactive protein (CRP) and serum amyloid P component (SAP) monoclonal antibodies with limulin and pentraxins of other species, Immunology 76(2) (1992)　324-30.
24. Nga Yen Nguyen, Akira Suzuki, Sheau-Mei Chang , Isolation and characterization of limulus C-reactive protain genes, J Biol.Chem.261 (1986) 10450-10455.
25. Ke-Jian Lei,Teresa Liu,Gerald Zon, Genomic DNA sequence for human C-reactive protein, J Biol.Chem. 260(1985) 13377-13383.