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INFLAMMATION AND IMMUNOPHARMACOLOGY

Critical Role of L-Selectin and Histamine H4 Receptor in Zymosan-Induced Neutrophil Recruitment from the Bone Marrow: Comparison with Carrageenan

Bayer Yakuhin, Ltd., Research Center Kyoto, Respiratory Disease Research, Kyoto, Japan

Received for publication December 2, 2003
Accepted February 27, 2004.

	Abstract

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Zymosan and carrageenan represent two inflammatory stimuli leading to significant neutrophilia when injected into mice. Despite several similarities between the two proinflammatory agents, the mechanisms leading to neutrophil influx into the site of stimulus injection are unclear. As demonstrated by antibody (Ab) studies directed against adhesion molecules, L-selectin was pivotal for zymosan-induced but not carrageenan-induced pleurisy. Zymosan but not carrageenan injection into the pleural cavity caused blood neutrophilia and significant release of neutrophils from the bone marrow, events that were inhibited by anti-L-selectin but not anti-Mac-1 Ab pretreatment. Pertussis toxin, known to regulate cell efflux, abrogated both zymosan- and carrageenan-induced pleurisy, but only zymosan-induced neutrophil release from the bone marrow. Dexamethasone, known to inhibit pleurisy induced by either stimulus, had no effect on bone marrow neutrophil numbers. The G_i/o G protein-coupled H₄ histamine receptor is highly expressed in the bone marrow and on leukocytes and plays an important role in zymosan-induced pleurisy in vivo. Zymosan-triggered neutrophil release from bone marrow was abrogated by pretreatment of mice with thioperamide, a known H_3/4 receptor antagonist, whereas H₁ and H₂ receptor antagonists had no effect. Moreover, histamine itself, when injected intravenously, led to a similar time- and dose-dependent decrease of neutrophil numbers in the bone marrow that was inhibited by thioperamide. Because the H₃ receptor is not expressed on neutrophils, these findings indicate that both H₄ and L-selectin regulate zymosan-induced neutrophil release from bone marrow and subsequent infiltration in the pleurisy model.

To experimentally induce leukocyte trafficking, carrageenan (sulfated polyanionic polysaccharide) and zymosan have become two commonly used inflammatory agents (Oh-ishi, 1997). Carrageenan-induced pleurisy is an experimental model of acute inflammation characterized by the migration of phagocytic cells. Polymorphonuclear leukocytes are the predominant cell type infiltrating the pleural cavity within the first 12 h after carrageenan injection. Later, polymorpho-nuclear cells disappear and are replaced by migrating mononuclear cells, which differentiate into macrophages and dominate the reaction up to its resolution at 48 h (Tomlinson et al., 1994; Willis et al., 1996). These inflammatory cells synthesize and release various mediators of inflammation, among which the kallirein-kinin system and prostaglandins play a pivotal role. Inhibitors of cyclooxygenase, kallikrein-kinin, and bradykinin are capable of blocking carrageenan-induced pleurisy in vivo (Katori et al., 1978; Dozen et al., 1989). Previous experiments have shown that neutrophil migration is reduced by agents capable of blocking the release of a specific neutrophil attractant from macrophages (Moraes et al., 1993). Likewise, macrophage depletion experiments reducing the resident macrophage population by about 80% significantly blocked neutrophil migration induced by carrageenan; however, such a treatment did not affect neutrophil migration induced by chemokines (Souza et al., 1988). Thus, these results support the suggestion that macrophages participate in the control of neutrophil migration induced by carrageenan, which is mediated by the kallikrein-kinin system and prostaglandins.

Zymosan, the insoluble polysaccharide component of the cell walls of Saccharomyces cerevisiae, is also commonly used for pleurisy induction in vivo. Like carrageenan, zymosan injection leads to the migration of phagocytic cells. Neutrophils are the predominant cells found in the pleural cavity up to 12 h after zymosan challenge, before they are replaced by macrophages that dominate the reaction at later stages (Oh-ishi, 1997). Despite these striking similarities in the leukocyte constitutions of the inflammatory exudates, the mechanisms leading to neutrophilia obviously differ between zymosan and carrageenan (Damas and Remacle-Volon, 1986; Vannier et al., 1989). Histamine, platelet-activating factor (PAF), the complement system, and LTs, but not prostaglandins, are the main mediators responsible for the cell influx into the pleural cavity in response to zymosan (Tarayre et al., 1989; Imai et al., 1991). Extensive in vitro studies indicate multiple inflammatory mechanisms of zymosan action, including the activation of the alternative complement activation pathway and the generation of the anaphylatoxins C3a and C5a, agents well known for their capacity to induce the release of histamine, PAF, and LT from mast cells. The latter play a critical role in initiating cell migration into the pleural cavity after zymosan injection. We recently demonstrated that mast cells and the H₄ receptor play an important role in zymosan-induced neutrophil migration into the pleural cavity. Based on the current understanding of the zymosan pathobiology, a cascade triggered by zymosan binding to toll-like receptor 2, which is expressed on mast cells, can induce mast cell activation (McCurdy et al., 2003) and mediate the zymosan signal in vivo via MyD88 (Takeshita et al., 2003).

We have undertaken experiments to determine mechanistic similarities and differences responsible for initiating the inflammatory infiltrates induced by zymosan and carrageenan and have shown that although both agents can release bone marrow neutrophils that are likely the source of the pleural infiltrate, the signaling and trafficking mechanisms responsible for this infiltration differ markedly.

	Materials and Methods

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Chemicals. Dexamethasone, zymosan, histamine, thioperamide, pyrilamine, and cimetidine were purchased from Sigma-Aldrich (St. Louis, MO). Carrageenan, wortmannin, and all bulk chemicals not further specified were purchased from Wako Pure Chemicals (Osaka, Japan). Anti-L-, E-, and P-selectin, anti-Mac-1, and anti-LFA-1 Ab and Ig-matched control Ab were purchased from BD Biosciences Pharmingen (San Diego, CA). Pertussis toxin (PTX) was purchased from Calbiochem (San Diego, CA).

Mice. Balb/c mice were purchased from Charles River Inc. (Yokohama, Japan). Animals were kept under standard conditions in a 12-h day/night rhythm with free access to food and water ad libitum. All animals received humane care, and the studies have been approved by the internal ethic committee in accordance with the guidelines recommended by the Japanese Association of Laboratory Animal Science.

Zymosan- and Carrageenan-Induced Pleurisy, Blood Neutrophilia, and Bone Marrow Neutrophil Release. Mice (Balb/c, female, 7–8 weeks old; Charles River Inc.) received a single intrapleural injection of 0.2 ml of PBS containing -carrageenan (500 µg/mouse) or zymosan (100 µg/mouse) under anesthetization with ether. Test compounds (PTX, Ab, Y-27632, wortmannin, and dexamethasone) and vehicle (PBS) were administered i.v. (0.2 ml/head) 3 min before carrageenan or zymosan. Four hours after injection, mice were euthanized, and pleural fluid was collected by washing the pleural cavity twice with 2 ml of PBS. The cell suspensions were diluted to one-tenth in Turk's stain solution (Nacalai Tesque, Kyoto, Japan). The number of total cells in the sample was counted under the microscope using a hemocytometer. Cytospin specimens were stained with May-Gruenwald's (Merck Biosciences, Darmstadt, Germany) and Giemsa's solution (Merck Biosciences) for leukocyte typing. The distribution of each cell population (neutrophils, eosinophils, macrophages, lymphocytes, and others) was counted under microscopy by counting 200 to 300 cells.

For assessment of neutrophilia and neutrophil release from bone marrow, similarly treated separate groups of mice were euthanized to obtain peripheral blood from the abdominal vein at the indicated time points after injection and then mice were completely bled. Immediately after the bleeding, the left femur was isolated and the femoral head and condyles were removed. The displaceable cells were recovered by flushing the lumen of the femur shaft with 1 ml of PBS. The cell suspension was diluted to one-tenth in Turk's stain solution, and cell differentials and counting were performed as described above.

Statistics. Data are expressed as means ± S.E.M. Statistical significance was determined using the unpaired Student's t test if applicable or results were analyzed by using one-way ANOVA, and if variances were nonhomogeneous differences between groups were assessed, by using Dunnett's method from commercially available statistic software (GraphPad Software Inc., San Diego, CA). Values of P < 0.05 were considered as statistically significant (^*P < 0.05, ^**P < 0.01).

	Results

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The coupling specificity of the G and G subunits of G_i/o proteins initiates signaling pathways triggered by chemokines and chemoattractants upon binding to their G protein-coupled receptors. Typically, G_i/o proteins are sensitive to inhibition by PTX, which inhibits cell migration induced by numerous stimuli. To compare the PTX sensitivity of leukocyte recruitment induced by zymosan and by carrageenan, respectively, we evaluated the effect of PTX on neutrophil influx into the pleural cavity of mice. PTX inhibited neutrophil migration triggered by zymosan and by carrageenan; however, the carrageenan model turned out to be more sensitive (90 versus 60% inhibition of the zymosan response) as shown in Fig. 1, A and B. Thus, both the chemotaxis inhibitor PTX and dexamethasone, the gold standard anti-inflammatory drug, qualitatively showed similar effects on zymosan- and carrageenan-induced inflammation in the mouse (cf. Fig. 1; Takeshita et al., 2003).

View larger version (26K): [in this window] [in a new window]   Fig. 1. Inhibition by PTX and effects of anti-selectin Ab on neutrophil migration into the pleural cavity induced by zymosan and carrageenan. Mice received 3 µg/kg PTX or the corresponding volume of PBS i.v. (0.2 ml/head) 3 min before zymosan (A) or carrageenan (B) injection into the pleural cavity. Four hours later, mice were euthanized, pleural fluid was collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Note the differences in the scale of the y-axes. Results were analyzed using Student's t test. ***, P < 0.001 versus the respective control group. Mice received 100 µg/kg anti-L (C), -E (D), -P (E), or the corresponding control Ab i.v. (0.2 ml/head) 3 min before zymosan or carrageenan injection into the pleural cavity. Four hours later, mice were euthanized, pleural fluid was collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Results were analyzed using Student's t test. **, P < 0.01 ***, P < 0.001 versus the respective control group.

To identify potential mechanistic differences between the two models of neutrophilia, we next evaluated the contribution of adhesion molecules to neutrophil influx into the pleural cavity induced by zymosan or carrageenan. In neutrophil trafficking, L-, P-, and E-selectins are crucial for the initial adhesion contact, and tight adhesion interactions involve members of both Ig-like and the integrin families of adhesion proteins. In particular, the 2 integrins, among which LFA-1 and Mac-1 are most abundantly expressed on neutrophils, are critical for firm adhesion of rolling neutrophils to the endothelial cell surface and transendothelial migration. The central role of these selectins and 2 integrins is documented in various models of inflammation; however, a complete picture of their individual contributions to neutrophilia induced by zymosan or carrageenan, respectively, is not yet available. Mice pretreated with 100 µg of anti-E-selectin Ab showed a significant reduction of neutrophilia in both models, whereas pretreatment of mice with an anti-P-selectin Ab had no effect. In contrast, pretreatment of mice with an Ab directed against L-selectin reduced the inflammatory response to zymosan only without showing any effect on neutrophil numbers in the pleural cavity exudate of mice challenged by carrageenan (Fig. 1, C–E). To further elucidate the contribution of adhesion molecules, we investigated the role of the 2 integrins. Clearly, their functionality is also a prerequisite to allow neutrophils to invade into tissue upon zymosan or carrageenan injection, because significant reduction (anti-LFA-1 Ab) or even complete abrogation (anti-Mac-1 Ab) was seen under antibody treatment (Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 2. Anti-2 integrin Ab abrogate neutrophils migration into the pleural cavity induced by zymosan or carrageenan. Mice received 100 µg per mouse of anti-LFA-1 (A), anti-MAc-1 (B), or the corresponding control Ab i.v. (0.2 ml/head) 3 min before zymosan or carrageenan injection into the pleural cavity. Four hours later, mice were euthanized, pleural fluid was collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Statistical differences were analyzed using Student's t test. **, P < 0.01; ***, P < 0.001 versus the respective control group.

In general, zymosan-induced pleurisy was more severe than carrageenan- or chemoattractant (N-formyl-L-methionyl-L-leucyl-L-phenylalanine or LTB₄)-induced pleurisy in mice (cf. differences in cell numbers in Figs. 1 and 2; data not shown). In both cases, however, the relative neutrophil populations and the target body compartment neutrophils infiltrating, i.e., the pleural cavity, were identical. We thus wondered whether potential sources neutrophils were recruited from would differ and measured neutrophil numbers in the peripheral blood and in the bone marrow after intrapleural injection of zymosan or carrageenan. As illustrated in Fig. 3A, zymosan injection led to a 2-fold increase in blood neutrophil counts 2 h after challenge. In parallel, neutrophil numbers in the bone marrow declined, identifying the bone marrow as, potentially, a major source of neutrophils in the zymosan model. Surprisingly, neutrophil numbers did neither change significantly in the circulation nor in the bone marrow after carrageenan injection (Fig. 3B). To investigate the potential link between adhesive function and the mechanism of neutrophil recruitment, we next evaluated the effect of anti-L-selectin and anti-Mac-1 Ab on zymosan-induced neutrophil release from the bone marrow. Neutralization of L-selectin but not of Mac-1 resulted in a highly significant retention of neutrophils in the bone marrow (Fig. 3, C and D). Pretreatment of mice with PTX also conferred protection against zymosan-induced neutrophil release from bone marrow (Fig. 3E). In contrast, no effect at all on bone marrow neutrophils was seen by dexamethasone at doses known to clearly inhibit zymosan-induced pleurisy (Fig. 3F) (Takeshita et al., 2003).

View larger version (25K): [in this window] [in a new window]   Fig. 3. Time course of zymosan- or carrageenan-induced changes in neutrophil numbers in blood and bone marrow and effect of anti-L-selectin Ab, anti-Mac-1 Ab, PTX, and dexamethasone on the zymosan-induced release of neutrophils from the bone marrow. Male Balb/c mice received zymosan (A, 100 µg/mouse) or carrageenan (B, 500 µg/mouse) by intrapleural injection (0.2 ml/cavity). At the time point indicated, mice were euthanized, blood or bone marrow was collected, and neutrophil numbers were determined microscopically (blood, open circles; bone marrow, full circles). Data are expressed as mean values ± S.E.M. of five animals per group. Mice received equal doses (100 µg/mouse) of anti-L-selectin (C), anti-Mac-1 (D), or control Ab. PTX (3 µg/mouse; E), dexamethasone (3 or 30 mg/kg; F), or the corresponding volume of PBS (0.2 ml/head) was administered i.v. 3 min before zymosan injection into the pleural cavity. Two hours later, mice were euthanized, cells in bone marrow was collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Statistical differences were analyzed using Student's t test. ***, P < 0.001 versus respective control group.

We recently demonstrated that zymosan induced pleurisy in a mast cell- and LTB₄-dependent manner (Takeshita et al., 2003), likely through the activation of the H₄ histamine receptor. Thus, we evaluated the contribution of H_1–4 receptors to the neutrophil release from bone marrow induced by zymosan. Mice pretreated with maximally tolerable doses of the histamine H₁ receptor antagonist pyrilamine (10 mg/kg i.v.) or with the H₂ receptor antagonist cimetidine (30 mg/kg i.v.) showed no change in neutrophil release. In contrast, pretreatment of mice with thioperamide, an H_3/4 antagonist, significantly reduced the release of neutrophils from the bone marrow in a dose-dependent manner (Fig. 4).

View larger version (29K): [in this window] [in a new window]   Fig. 4. Prevention of zymosan-induced release of neutrophils from the bone marrow by the H4 antagonist thioperamide. Mice received various doses of the histamine receptor antagonists indicated or the corresponding volume of vehicle (V; 0.2 ml/head i.v.) 3 min before zymosan injection (100 µg/mouse) into the pleural cavity. Two hours later, mice were euthanized, bone marrow cells were collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Statistical differences were analyzed by using one-way ANOVA, and differences between groups were assessed using Dunnett's method (*, P < 0.05; **, P < 0.01). Statistical differences were analyzed using Student's t test where appropriate (H1 and H2).

We next wondered whether histamine itself could mimic zymosan-induced neutrophil recruitment and applied various doses of histamine i.v. and monitored the changes in leukocyte numbers in the circulation (data not shown) and in the bone marrow. Substantial neutrophil release from the bone marrow was observed within 1 h after challenge, and the dose-response relationship reached a maximum at 60 to 600 µg of histamine per mouse. Neutrophil release from bone marrow induced by a histamine dose of 300 µg/mouse peaked at 2 h after injection (Fig. 5B). Pretreatment of mice with thioperamide significantly reduced the histamine-induced release from bone marrow at all doses tested (Fig. 5E). Interestingly, and perhaps not surprisingly, the potency of thioperamide in counteracting the release of neutrophils from bone marrow was stronger against histamine compared with zymosan. Again, the H₁ receptor antagonist pyrilamine (10 mg/kg i.v.) and the H₂ receptor antagonist cimetidine (30 mg/kg i.v.) had no effect (Fig. 5, C and D).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Dose-response and time course of histamine-induced release of neutrophils from the bone marrow of the mouse and effects of histamine receptor antagonists. Male Balb/c mice received histamine by intravenous injection (0.2 ml/mouse) at the doses indicated (A) or at a dose of 300 µg (B). Two hours later (A) or at the time points indicated (B), mice were euthanized, bone marrow cells were collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Results were analyzed using one-way ANOVA, and differences between groups were assessed using Dunnett's method (*, P < 0.05; **, P < 0.01). Mice received various doses of the histamine receptor antagonists as indicated (C–E) or the corresponding volume of vehicle (V; 0.2 ml/head i.v.) 3 min before histamine injection into the abdominal vein. Two hours later, mice were euthanized, bone marrow cells were collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Statistical differences were analyzed by using one-way ANOVA, and differences between groups were assessed using Dunnett's method (*, P < 0.05; **, P < 0.01). Statistical differences were analyzed using student's t test where appropriate (H1 and H2).

To further substantiate the evidence for the G_i/o-coupled H₄ receptor being the mediator of histamine-induced neutrophil recruitment, we used inhibitors of signal transduction cascades known to be stimulated by histamine/H₄ receptor ligation and neutrophil migration. As expected, pretreatment of mice with PTX, the phosphatidylinositol 3-kinase inhibitor wortmannin, or the Rho kinase inhibitor Y-27632 decreased the number of neutrophils in the release from the bone marrow after histamine injection. Neutrophil release from the bone marrow, however, remained unchanged upon dexamethasone pretreatment (Fig. 6).

View larger version (25K): [in this window] [in a new window]   Fig. 6. Inhibition of histamine-induced neutrophil recruitment by PTX, wortmannin, or Y-27632. Mice received an i.v. injection of PTX (3 µg/mouse; A), wortmannin (WM, 3 mg/kg; B), Y-27632 (5 mg/kg; C), dexamethasone (30 mg/kg; D), or the corresponding volume of vehicle (PBS, 0.2 ml/head, V) 3 min before histamine application (300 µg/mouse i.v.). Two hours later, mice were euthanized, bone marrow cells were collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Statistical differences were analyzed using Student's t test. **, P < 0.01; ***, P < 0.001 versus respective vehicle-treated control group.

Finally, we wanted to further characterize and compare the molecular effectors involved in histamine-induced leukocyte trafficking with the zymosan-induced pleurisy and examined the effect of Ab directed against selectins. As shown in Fig. 7A, mice pretreated with L-selectin Ab did not exhibit neutrophilia, and the numbers of neutrophils in the bone marrow remained unchanged compared with nonhistamine-challenged controls. This resistance was highly specific for L-selectin, because Ab against E- and P-selectin failed to prevent the decrease of neutrophil numbers in the bone marrow after histamine injection (Fig. 7, B and C). In stark contrast to the zymosan-induced bone marrow efflux, however, anti-Mac-1 was effective in further promoting cell efflux (Fig. 7D).

View larger version (23K): [in this window] [in a new window]   Fig. 7. Effect of anti-selectin and anti-2 integrin Ab on histamine-induced release of neutrophils from the bone marrow. Mice received 100 µg/kg anti-L-(A), -E (B), or -P (C) selectin Ab or anti-Mac-1 (D), or the corresponding volume of the respective isotype-matched control Ab in PBS i.v. (0.2 ml/head) 3 min before histamine injection into the abdominal vein. Two hours later, mice were euthanized, bone marrow cells were collected, and the number of neutrophils was determined microscopically. Data are expressed as mean values ± S.E.M. of five animals per group. Statistical differences were analyzed using Student's t test. **, P < 0.01; ***, P < 0.001 versus respective control group.

	Discussion

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An important finding of these studies highlighting a fundamental difference between zymosan- and carrageenan-induced pleurisy was the observation that zymosan but not carrageenan led to neutrophil recruitment from the bone marrow. This might explain why the number of neutrophils in the pleural cavity induced by zymosan is 2- to 3-fold higher compared with carrageenan. Although 5-bromo-2-deoxyuridine pulse labeling experiments have not been performed to specifically determine compartmental contributions for either stimulus, carrageenan most likely triggers neutrophil recruitment from the circulating pool in the bloodstream or other tissue compartments. It has also been demonstrated that bone marrow-derived neutrophils preferentially sequester to the microvasculature of the lung (Lawrence et al., 1996; van Eeden et al., 1997b). The mechanistic uniqueness of zymosan seems to be mediated by L-selectin, mast cells (Takeshita et al., 2003), and probably other mast cell-derived products such as leukotriene B₄ and PAF. Furthermore, our Ab and pharmacological experiments show that neutrophil release from the bone marrow is dependent on functional G_i/o signaling, most likely triggered by histamine-H₄ receptor interaction, whereas it is insensitive to Mac-1 blockade and to steroids.

Physiologically, L-selectin expression is low in the mitotic pool of neutrophils, increases as cells mature in the postmitotic pool of the bone marrow, and is constitutively expressed on circulating neutrophils (van Eeden et al., 1995, 1997c). Importantly, neutrophils that are released from the bone marrow by inflammatory stimuli express higher levels of L-selectin compared with their circulating counterparts, and they progressively lose those L-selectin molecules as they age in the circulation (van Eeden et al., 1997a). It was reported that glucocorticoids decrease L-selectin expression on circulating neutrophils by down-regulating L-selectin expression in the maturation pool of the bone marrow (Nakagawa et al., 1999). Nevertheless, dexamethasone did not affect zymosan-induced neutrophil release from the bone marrow but dose dependently inhibited their extravasation into the pleural cavity (Fig. 3; Takeshita et al., 2003). Given the plurality of anti-inflammatory effects for which dexamethasone may be responsible, it is difficult to simplistically conclude a role in any one compartment using this inhibitor. Zymosan therefore probably increases the adhesion signal of L-selectin rather than L-selectin expression. However two important caveats must be taken into account. In mice, there is at least one report showing that carrageenan injected into the pleural cavity results not only in acute pleurisy but also increased neutrophil infiltration into the lung parenchyma (Cuzzocrea et al., 2003). There is also a suggestion in rats (Doherty et al., 1995) that zymosan injection causes plasma leakage and leukocyte extravasation from "milky spots" on the parietal pleural surface. Hence, the source of emigrating neutrophils may be profoundly different in the two models. This could account in part for the differential requirement for L-selectin between the two stimuli, according to the neutrophil source and the necessity for neutrophil rolling as a mechanism for efflux.

Emigrated neutrophils must move through connective tissue and then through the mesothelial lining before gaining entry into the pleural cavity. The profound inability of anti-Mac-1 antibody to affect neutrophil emigration from the bone marrow in response to zymosan only is therefore most intriguing given that the requirement for Mac-1 has been shown for neutrophil migration (basal to apical direction) across gut epithelium (Parkos et al., 1991). Nonetheless, the lack of, or lower Mac-1 expression in bone marrow compared with inflammatory exudates may account for the lack of effect by Mac-1 antibody on bone marrow extravasation as opposed to the infiltration to the pleural cavity. In the case of histamine-induced bone marrow efflux, it would seem that inhibition of Mac-1 promoted the efflux. It has been shown that histamine prevents complement fragment (C3bi)-induced Mac-1 clustering and chemoattractant-induced up-regulation and cell spreading in vitro, the suggestion being that histamine is negatively regulating cell adhesion (Francis et al., 1991). If a similar scenario occurs in vivo in the bone marrow whereby histamine inhibits Mac-1 function, explaining the result using anti-Mac-1 antibody becomes difficult. However, in the current experiments, no other factor has been characterized as promoting the migratory response of the bone marrow neutrophils; thus, a characterization of the direct effect of histamine on bone marrow neutrophil Mac-1 expression and functionality may provide clues to the mechanism involved. Even if histamine was negatively regulating bone marrow neutrophil adhesion through Mac-1, such a phenotype does not rule out the promotion of cell efflux through utilization of other adhesion receptors, including L-selectin. Additionally, although complement or chemoattractant-induced Mac-1 clustering and up-regulation are relevant to the firm adhesive state and migration capacity, antibody inhibition of the basal adherence may release sufficient numbers of cells to the pool available for efflux. Histamine may then stimulate efflux through activation of L-selectin or other adhesion receptor pairs. A more thorough analysis of Mac-1 expression on bone marrow neutrophils and the effect of cell activation is therefore warranted to understand the effect of the anti-Mac-1 antibody.

That the zymosan model turned out to be less sensitive (60 versus 90% inhibition of the response to carrageenan) to PTX might be due to PAF, which is released by zymosan- but not by carrageenan-stimulated mast cells. In contrast to other mast cell-derived chemotactic factors such as C5a, LTB₄, or prostaglandin D₂, which all signal through PTX-sensitive G proteins, PAF activation of eosinophils can activate a protein kinase C-driven pathway independent of G_i (Teixeira et al., 1997). It can be speculated that a similar signaling pathway is dominant in mast cells. Indeed, it has been demonstrated that pleurisy induced by zymosan, but not by carrageenan, was significantly reduced in PAF-desensitized or PAF receptor antagonisttreated animals (Martins et al., 1989).

Clearly, the results obtained with thioperamide, an H_3/4 antagonist, strongly argue for H₄ receptor control of neutrophil release from the bone marrow. The fact that histamine injection closely mimicked the effects of zymosan and that thioperamide was the only histamine receptor antagonist able to block neutrophil recruitment shows that histamine is an endogenous mediator of zymosan-induced pleurisy and also that H₄ receptor activation is pivotal for this to happen. Interestingly, however, the inhibitory actions of thioperamide, or, for instance, PTX, to histamine-induced decreases in neutrophil numbers in the bone marrow were not complete. The absolute numbers of neutrophils harvested from the bone marrow of nontreated mice averaged 16 to 20 million as illustrated in Fig. 2 and dropped to 2.5 to 5 million after zymosan or histamine application. Thioperamide or PTX pretreatment resulted in a "rescue" of 60 to 70% of neutrophils, i.e., the numbers of neutrophils in the bone marrow were brought back to 10 to 12 million under drug treatment. The question of why the rescue was not complete is difficult to answer. The most likely explanations, however, are that 1) thioperamide and/or PTX did not effectively reach all target receptor molecules activated by histamine; 2) pharmacokinetic properties of the test compounds would not allow for full efficacy; or 3) an additional, non-G_i/o-coupled, thioperamide-insensitive histamine receptor or histamine-induced biological response plays a role. There is, however, no room for a contribution of H₁ or H₂ receptors, because even at very high doses pyrilamine and cimetidine were without any effect. By using thioperamide as a tool to study H₄ biology, a possible role of H₃ cannot be completely ruled out; however, it is rather unlikely given the completely different expression pattern of H₃ compared with H₄, the former being absent on most if not all leukocytes (Gantner et al., 2002). Furthermore, a contribution of H₃ would not explain the lack of full efficacy by thioperamide and PTX in this model. However, in vitro studies performed with histamine-stimulated neutrophils from humans, mice, and rats were all negative with regard to chemotaxis induction (data not shown); therefore, it is still unclear whether histamine induces neutrophil migration directly via H₄ activation in vivo. Histamine can lead to the up-regulation of P-selectin, an endothelial cell adhesion glycoprotein expressed early on during an inflammatory process on the cell surface where it binds to blood leukocytes (Burns et al., 1999). This histamine effect, however, is mediated by the H₁ receptor (Weber et al., 1997). Because pyrilamine failed to influence histamine-induced neutrophil recruitment in our model (cf. Fig. 4), and P-selectin antibody did not seem to inhibit the response, it is unlikely that this is a plausible explanation. In neutrophils, activation of L-selectin induces a variety of responses, including calcium flux, activation of the respiratory burst, and, importantly, potentiation of G_i/o signaling (Hornquist et al., 1997), which ultimately could explain how H₄ receptor- and L-selectin-derived signals cooperate and then impact the more "global" migration-inducing signaling events such as activation of phosphatidylinositol 3-kinase and Rho (Fig. 6). L-selectin cross-linking induces tyrosine phosphorylation as well as activation of mitogen-activated protein kinases, initiating in turn a signaling cascade involving L-selectin phosphorylation, recruitment of the signaling molecules Grb2/Sos, and activation of Ras and Rac2 (Ebnet and Vestweber, 1999; Patel et al., 2002). Thereby, L-selectin and H₄ signals might synergistically trigger migration in neutrophils in vivo. Putting our findings and the published observations together, the remaining unanswered question is how histamine influences the L-selectin system. It can be hypothesized that the sensitivity of the L-selectin system toward activation increases. Possibly, histamine influences the expression levels of L-selectin and/or its ligands in the bone marrow, affects the avidity of L-selectin to their ligand(s), or a combination of both.

Collectively, the similarities and differences between neutrophilia induced by zymosan and carragenan, respectively, can be summarized as follows. Both models depend on E-selectin, Mac-1, LFA-1, and G_i/o signaling, are sensitive to dexamethasone (Takeshita et al., 2003), partly depend on tumor necrosis factor- (unpublished observation), and do not depend on P-selectin. The models clearly differ with regard to the necessity of mast cells, L-selectin, histamine, and its action on H₄ receptor, and the number of cells recruited to the site of injection and the body compartment of neutrophil origin (Takeshita et al., 2003; this study). Finally, their sensitivity to inhibitors of G_i/o proteins differs (carrageenan more sensitive; cf. Fig. 1), lipoxygenase (zymosan more sensitive; Rao et al., 1994), cyclooxygenase (carrageenan more sensitive; Calhoun et al., 1987), and PAF antagonists (zymosan more sensitive; Martins et al., 1989).

In using these models to evaluate the efficacy of anti-inflammatory compounds, it is important to bear in mind that, at face value, these two models simply facilitate a general assessment of in vivo trafficking of leukocytes. However, it will be critical to carefully assess their comparability and relevance to the mechanistic intricacies of the physiological process being antagonized, as well as the specific compartments being examined. Given the complexity of leukocyte immune surveillance, trafficking, and recruitment, as well as the highly variable kinetics of mediator release and duration of action, a more detailed assessment of the source of recruited leukocytes may have significant impact on the evaluation of the efficacy of a particular therapeutic.

	Footnotes

 
DOI: 10.1124/jpet.103.063776.

ABBREVIATIONS: PAF, platelet-activating factor; Ab, antibody; PTX, pertussis toxin; ANOVA, analysis of variance; LT, leukotriene; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide..

Address correspondence to: Dr. Kevin B. Bacon, Bayer Yakuhin, Ltd., Research Center Kyoto, Respiratory Diseases Research, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, 619-0216 Kyoto, Japan. E-mail: kevin.bacon.kb{at}bayer.co.jp

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