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The Journal of Immunology, 1998, 161: 3631-3638.
Copyright © 1998 by The American Association of Immunologists

Inhibition of Leukocyte Emigration Induced During the Systemic Inflammatory Reaction In Vivo Is Not Due to IL-81

Boris Schleiffenbaum2,*, Jörg Fehr*, Bernhard Odermatt{dagger} and Roland Sperb*

* Division of Hematology, Department of Internal Medicine, and {dagger} Department of Pathology, University Hospital Zürich, Zürich, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In keeping with the multistep model of leukocyte-endothelial cell interaction, stimulation of endothelium by cytokines or endotoxin (LPS) in vitro leads to selectin/integrin-mediated neutrophil adhesion, followed by neutrophil endothelial transmigration. The i.p. injection of LPS in vivo induces a systemic inflammatory reaction in a mouse model with generalized activation of both endothelial cells (up-regulation of adhesion molecules ICAM-1, VCAM-1, E-selectin) and neutrophils (up-regulation of Mac-1). However, no intravascular endothelial adhesion or tissue emigration of neutrophils can be observed. Even more importantly, the in vivo emigration of polymorphonuclear cells at sites of a local inflammatory reaction (IL-8, TNF, LPS) is totally inhibited when the mice are pretreated systemically with LPS, although the neutrophils respond fully to a rechallenge with LPS ex vivo, and endothelial adhesion molecules are further up-regulated locally. The systemic application of TNF also caused a total inhibition of neutrophil emigration. However, while anti-TNF mAb abrogated the inhibitory activity induced by TNF, they had no effect on systemic LPS. The systemic application of IL-8 did not inhibit neutrophil emigration, nor did the pretreatment of mice with anti-IL-8 mAb before the systemic application of LPS abrogate the inhibitory activity induced by LPS. Therefore, the putative inhibitor of neutrophil emigration, which may be of great physiologic importance, as it prevents in vivo the generalized emigration of activated neutrophils, most likely is not IL-8.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Results of experiments that use adhesion molecule-directed mAbs or recombinant adhesion receptors in vitro and in vivo have led to a model of ordered sequential leukocyte-endothelial cell interactions as the basis of leukocyte emigration through endothelium. Initial tethering of leukocytes to the vessel wall by a first class of adhesion molecules, selectins, results under the forces of flowing blood in tethering and rolling of leukocytes along the vessel wall. At some point, a rolling leukocyte may become attached and activated by cytokines, chemokines, or chemoattractants, and possibly directly by the interaction of adhesion molecules, leading subsequently to firm leukocyte adhesion and spreading on endothelium. Activation with respect to adhesion includes an increase in the affinity of a second class of adhesion molecules, integrins, for their ligands, redistribution of integrin receptors, and interaction of integrins with cytoskeletal proteins. Firm adhesion of leukocytes on the endothelial cell is followed by migration of the leukocyte to the endothelial intercellular junctions, diapedesis through the intercellular junction, and finally, migration into the subendothelial tissue (1).

In keeping with this multistep model of leukocyte-endothelial cell interaction, stimulation of endothelium by various cytokines or endotoxin (LPS) in vitro leads to selectin/selectin-counter-receptor-mediated tethering and rolling of neutrophils (PMN)3 on the endothelial monolayer under shear stress, followed by integrin/integrin-receptor-mediated attachment and definite neutrophil adhesion. Subsequent transmigration of neutrophils through the endothelial monolayer is dependent mostly on integrins and their counter-receptors ICAM-1 and ICAM-2 of the Ig superfamily (1). The most important players in neutrophil adhesion and endothelial transmigration in this orchestra of adhesion molecules are L-selectin (CD62-L, expressed on unstimulated PMN, counter-receptor on activated human endothelial cells unknown) (2, 3, 4, 5, 6), E-selectin (CD62-E, expressed on activated endothelium, counter-receptor on PMN still a matter of debate) (6, 7, 8), and P-selectin (CD62-P, expressed on unstimulated endothelium; up-regulated on the endothelial cell surface from intracellular Weibel-Palade bodies and by de novo protein synthesis, counter-receptor sialyl Lewis x (sLex)/P-selectin glycoprotein ligand-1 (PSGL-1) on PMN) (6, 7), which are responsible for PMN tethering and rolling on endothelium, and the ß2-integrins Mac-1 (CD11b/CD18; counter-receptor ICAM-1, fibrinogen, C3bi, and probably others on endothelium) (9, 10, 11) and LFA-1 (CD11a/CD18; counter-receptor ICAM-1, ICAM-2 on endothelium) (10, 11), which mediate, when activated, neutrophil firm attachment and adhesion as well as transmigration (1). Although it has been shown that an active participation of the endothelial cell is necessary for leukocyte migration (12), it is not clear whether neutrophil migration on cytokine-stimulated endothelium occurs along a chemotactic gradient or as haptotaxis toward the highest concentration of adhesion receptor ligands on the endothelial cell (13). In vitro, LPS or cytokine stimulation of endothelial cell monolayers alone is sufficient not only to induce neutrophil tethering and adhesion on endothelium (4, 14), but also to initiate unidirectional migration of neutrophils through the endothelial monolayer (11, 15). Therefore, it has been assumed that the secretion of an endothelial cell-derived chemokine such as IL-8 by activated endothelial cells together with the up-regulation and interaction of adhesion molecules on both neutrophils and endothelial cells could well explain neutrophil endothelial cell transmigration (16, 17).

However, in sepsis, a clinical in vivo model of systemic inflammation with exposure of endothelial cells of all organs to elevated plasma levels of LPS, a generalized tissue emigration of neutrophils has not been noted (18). We therefore speculated that neutrophil emigration in vivo either depends on additional factors other than the adhesion molecules and cytokines/chemokines known to regulate transmigration of neutrophils in vitro, or that, alternatively, inhibitors of neutrophil emigration exist in vivo that are not active in the in vitro test systems. In an animal mouse model, we can demonstrate that the systemic stimulation with LPS in vivo leads to activation of both the endothelium and neutrophils, but is not sufficient to cause generalized emigration of neutrophils into tissue. Furthermore, neutrophil emigration into sites of local inflammation induced by a second injection of LPS into a rear footpad is totally inhibited in mice that have been pretreated systemically with LPS. The inhibition of neutrophil emigration into sites of local inflammation that was observed in all control animals argues against the necessity of additional adhesion molecules or cytokines for the in vivo emigration of neutrophils and favors the existence of an inhibitor of neutrophil emigration. This inhibitor of neutrophil emigration is not active during the in vitro incubation of endothelium with LPS, but is induced in vivo by the systemic exposure to LPS. We provide evidence that IL-8, which has been shown to inhibit neutrophil transmigration in vitro and in vivo, is not the inhibitor induced by the systemic application of LPS in mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse experiments

Six- to eight-week-old SV-129 mice (Biologic Research Laboratories, Füllinsdorf, Switzerland), which were chosen for their susceptibility to LPS and the quality of leukocyte differentiation in forward/sideward scatter graphs of laser beam deflection in fluorescence flow cytometry, were injected i.p., either with LPS (R595, 10 µg (Sebak, Aldenbach, Germany) in 200 µl and 154 mM NaCl), mouse rTNF (Genzyme Diagnostics, Cambridge, MA; at various concentrations, in 200 µl and 154 mM NaCl), IL-8 (Genzyme Diagnostics; 0.625–5 µg, in 200 µl and 154 mM NaCl), or carrier solution only (200 µl and 154 mM NaCl). The doses of LPS, TNF, and IL-8 were chosen, as they induced a pronounced systemic inflammatory reaction (transient neutropenia, up-regulation of adhesion molecules), yet did not cause changes in animal behavior or appearance (>=100 µg LPS) and lay well below the LPS doses that proved to be fatal to individual animals in initial dose-finding studies (>=1500 µg LPS). After 2 h, mice were injected into the right rear footpad with either LPS (R595, 0.1 µg, in 50 µl and 154 mM NaCl), TNF (50 ng, in 50 µl and 154 mM NaCl), or IL-8 (5 ng, in 50 µl and 154 mM NaCl), and with carrier solution only into the left rear footpad. At the chosen dosage, the local application of LPS, TNF, or IL-8 led to a discernible swelling of the rear footpads within 4 h without causing too much discomfort to the animals (no limping or changes in behavior). In some experiments, the animals were pretreated with polyclonal rabbit anti-mouse TNF antiserum (400 µl i.p.; Genzyme Diagnostics) or anti-IL-8 mAb (DM/C7, 0.25 mg in 200 µl NaCl, 154 mM; Genzyme Diagnostics) (19, 20). Blood (100 µl) derived from the animals by tail vein incision was taken before the initial i.p. LPS injection and at the indicated time points, and was always immediately diluted (1/10 in PBS) and placed on ice. A sample (50 µl, diluted 1/2) was used to perform the white blood count (H1, Technicon) and to prepare blood smears for later microscopic leukocyte differentiation (Wright-Giemsa stain). The remaining 50 µl of blood (1/10 in PBS) was left on ice until stained by immunofluorescence technique (see below). The animals were sacrificed as indicated, and tissue sections were taken for later morphologic and immunohistologic work-up.

In some cases, rIL-8 (50 µg/kg, i.p.; Ser-IL-877; Genzyme Diagnostics) was given to the mice systemically instead of LPS. The i.p. injection had to be repeated at 90 min and at 180 min due to the short t1/2 of the cytokine to yield plasma concentrations >10 ng/ml throughout the experiments (determined in pilot studies). Again, LPS was injected into the right rear footpad of the animals 120 min after the initial injection of rIL-8 i.p., with the injection of carrier solution only into the left rear footpad serving as control. The animals were sacrificed 250, 275, 300, 325, and 350 min after systemic administration of rIL-8 i.p., and the rear footpads were taken for histologic and immunohistologic work-up. All animal experiments were performed according to the regulations of the Animal Care and Use Committee of the Canton of Zurich and were approved by the committee.

Immunofluorescence and flow cytometry

Immunophenotyping was performed with some modifications, as previously published (9). In brief, staining was performed in heparinized PBS-diluted whole blood (100 µl) at 4°C to avoid any alterations in Ag expression that might have been induced by leukocyte separation over Ficoll or dextran (21, 22). Leukocytes were stained by fluorochrome-conjugated mAbs, as indicated (15 min, 4°C). When unconjugated primary Abs were used, cells were washed twice after primary Ab incubation and reincubated with FITC-conjugated rabbit anti-rat polyclonal secondary Ab (15 min, 4°C; indirect immunofluorescence). Samples were cleared of red cells by hypotonic lysis (aqua tri-distillate). Single cell fluorescence corresponding to cell surface Ag expression was determined on a FACScan cytofluorometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The neutrophils (polymorphonuclear leukocytes, PMN), monocytes, lymphocytes, and, when present, eosinophils could clearly be differentiated by their forward and sideward scatter characteristics. Forward/sideward scatter gating was verified by differing density of CD45 expression on eosinophils and neutrophils, and monocytes and lymphocytes, and by the differential expression of Mac-1 on neutrophils and lymphocytes. Mean fluorescence, as measured in linear mode, was taken as a measure of cell surface Ag density and expressed in normalized artificial units with the fluorescence of cells of negative controls arbitrarily set at 50.

To examine the ex vivo responsiveness of neutrophils toward LPS activation at all time points, a sample of diluted whole blood (50 µl) was incubated in vitro with LPS (50 ng/ml, 30 min, 37°C), and changes in the expression of cell surface Ags were measured by flow cytometry. The rat anti-mouse mAbs I3/2 (rat anti-murine CD45; Sigma), M1/70 (rat anti-mouse Mac-1/LY-40; Serotec, Oxford, U.K.), and Mel-14 (rat anti-mouse L-selectin, IgG2a; American Type Culture Collection (ATCC), Manassas, VA) were used either unconjungated or as phycoerythrin conjugates (CD45). All unconjugated primary Abs were detected with rabbit anti-rat FITC-conjugated secondary polyclonal Abs (Serotec) in indirect immunofluorescence technique, as described.

Histology and immunohistology

Tissue sections from kidney, lung, and spleen, as well as both rear footpads were either fixed and embedded in methacrylat for morphologic evaluation or snap frozen in liquid nitrogen for immunohistologic staining. Tissue sections of 5 µm thickness were cut in a cryostat, placed on siliconized glass slides, air dried, fixed with acetone for 10 min, and stored at -70°C. Rehydrated tissue sections were incubated with the following rat primary mAbs: anti-Mac-1 (M1/70; Serotec, Oxford, U.K.), anti-ICAM-1 (KAT-1; R&D Systems, Abingdon, U.K.), anti-E-selectin (10E9.6; courtesy of Dr. D. Vestweber, University of Münster, Münster, Germany), anti-L-selectin (Mel-14, IgG2a; ATCC), and anti-VCAM-1 (M/K-2; Serotec). Primary rat Abs were revealed by sequential incubation rabbit Abs against rat Igs and rat alkaline phosphatase anti-alkaline phosphatase complexes (Dako, Glostup, Denmark). For the detection of E-selectin and L-selectin, this procedure was repeated twice. Secondary affinity-purified polyclonal anti-Ig antisera were diluted in Tris-buffered saline (TBS, pH 7.4) containing 5% normal mouse serum. All other dilutions were made in TBS alone. Incubations were done at room temperature for 30 min; TBS was used for all washing steps. Alkaline phosphatase was visualized using naphtol AS-BI phosphate and New Fuchsin as substrate, which yields a red precipitate. Endogenous alkaline phosphatase was blocked by levamisole. Color reactions were performed at room temperature for 15 min with reagents from Sigma. Sections were counterstained with hemalaum, and coverslips were mounted with glycerol/gelatin.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The i.p. application of LPS causes a systemic inflammatory reaction and a generalized up-regulation of adhesion molecules on both neutrophils and endothelium

Mice injected with 10 µg LPS (i.p.) showed a pronounced but transient neutropenia as part of their systemic inflammatory reaction. PMN counts began to drop at approximately 15 min and returned to baseline values approximately 4 h after the i.p. injection of LPS (Fig. 1GoA). The systemic application of LPS also caused a strong up-regulation of Mac-1 cell surface expression on neutrophils that lasted throughout the observation period (7 h; Fig. 1GoB). Surprisingly and in contrast to all in vitro observations, the in vivo stimulation of PMN by LPS or secondarily induced cytokines in vivo resulted only in a slight decrease of cellular L-selectin (about 40%) similar to unstimulated controls (Fig. 1GoC). Subpopulation effects caused by the influx of fresh neutrophils from the bone marrow, intravascular margination, or tissue emigration of PMN do not explain these unexpected findings, as the very same cells that failed to shed L-selectin had fully up-regulated their Mac-1 surface expression following stimulation by LPS and/or secondarily induced cytokines in vivo (Fig. 1GoB). Similarly, the exposure to systemic LPS led to a generalized stimulation of vascular endothelium and the up-regulation of endothelial adhesion molecules ICAM-1, VCAM-1, and E-selectin (Fig. 2Go; Table IGo). While ICAM-1 is expressed on resting endothelial cells of both small venules and capillaries, VCAM-1 is only expressed on resting small venule endothelium, but not on capillary endothelial cells, and E-selectin is not at all expressed on unstimulated endothelial cells in SV-129 mice in vivo. The systemic inflammatory reaction following the i.p. injection of LPS leads to the up-regulation of the endothelial adhesion molecules, which was first noted at 2 h (not shown) and most pronounced at 5 to 7 h after the LPS injection.



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  		FIGURE 1. Time response of peripheral neutrophil counts and neutrophil cell surface expression of Mac-1 and L-selectin on neutrophils. SV-129 mice were injected with 10 µg LPS in 200 µl and 154 mM NaCl i.p. (solid line), or 200 µl and 154 mM NaCl i.p. alone (dashed line). After 2 h, all mice were injected into the right rear footpad with LPS (0.1 µg in 50 µl and 154 mM NaCl), and with carrier solution only into the left rear footpad. Blood was taken as indicated, and white blood counts were done on a H1 Technicon cell counter, and white cell differentials were performed on Wright-Giemsa-stained blood smears (A). Mac-1 (M1/70, rat anti-mouse mAb, B) and L-selectin (Mel-14, rat anti-mouse mAb, C) expression were determined on peripheral neutrophils stained by indirect immunofluorescent technique (flow cytometry, FACScan). PMN of mice treated systemically with LPS were reexposed to LPS (50 ng/ml) ex vivo (bar, C).

 


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  		FIGURE 2. Expression of endothelial adhesion molecules in correlation to neutrophil emigration in systemically stimulated and unstimulated mice. SV-129 mice were injected with 10 µg LPS in 200 µl and 154 mM NaCl i.p. (F–J) or 200 µl and 154 mM NaCl i.p. (A–E) alone. After 2 h, all mice were injected into the right rear footpad with LPS (0.1 µg in 50 µl and 154 mM NaCl), and with carrier solution only into the left rear footpad. The animals were sacrificed at various time points (see Materials and Methods and Table IGo; animals sacrificed at 300 min are shown in the figure). Immunohistology was done on frozen sections of kidney (A and F, with some background staining present on renal tubuli), left rear feet (B and G), and right rear feet (C and H) using standard indirect staining techniques and anti-E-selectin mAbs (10E9.6; courtesy of Dr. D. Vestweber). In addition, Wright-Giemsa-stained thin sections of methacrylat-embedded right rear feet were examined by light microscopy (D and I, x125; E and J, x783). Other organs, such as spleen and lung, were also examined, and showed results similar to those found in kidney. The expression of adhesion molecules ICAM-1 (higher baseline expression) and VCAM-1 took a similar time course (see Table IGo). The figure is representative of three independent experiments, with similar results obtained at other time points up to 430 min (see Table IGo).

 

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  		Table I. Kinetics of endothelial adhesion molecule expression following systemic stimulation with LPS1

 
In contrast to in vitro experiments, systemic exposure of neutrophils and endothelial cells to LPS and subsequent up-regulation of neutrophil and endothelial adhesion molecules do not lead to generalized tissue emigration of neutrophils in vivo

The i.p. injection of 10 µg LPS into mice (25–30 g total body weight) leads to LPS plasma concentrations of LPS that are more than sufficient to cause up-regulation of endothelial adhesion molecules and induction of PMN transmigration in vitro (maximum 80 ng/ml). In vivo, however, although murine neutrophils were activated (up-regulation of Mac-1) and all vascular endothelium had up-regulated their adhesion molecules after systemic exposure to LPS (Fig. 2GoF, immunohistology of kidney; Table IGo, immunohistology of lung, kidney, and left footpad), a generalized tissue emigration of PMN or other leukocytes that would have been expected, based solely on the cited in vitro experiments, was not noted (Fig. 2GoF, immunohistology of kidney; Table IIGo, left footpad, and results not shown). Despite strong expression of ICAM-1, VCAM-1, and E-selectin on vascular endothelium of all organs tested, neutrophils did not adhere to activated vascular endothelium, and no neutrophils could be detected in the surrounding tissues.


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  		Table II. Emigration of PMN at sites of local inflammation1

 
The systemic inflammatory reaction following the i.p. injection of LPS induces inhibition of neutrophil emigration

The injection of LPS (0.1 µg) into the rear footpad of mice leads to local up-regulation of endothelial adhesion molecules ICAM-1, VCAM-1, and E-selectin (Fig. 2GoC, Table IGo, right footpad) and a marked tissue infiltration of neutrophils starting approximately 2 h after the injection (Fig. 2Go, C, D, and E, Table IIGo, right footpad). However, when mice were pretreated with LPS systemically (10 µg LPS i.p., 120 min before the local injection of 0.1 µg LPS), PMN emigration was totally inhibited (Fig. 2Go, H, I, and J, Table IIGo, right footpad). Even more importantly, the careful revision of all slides prepared from the rear footpads locally injected with LPS at different depths did not reveal adhesion of PMN to vascular endothelial cells in mice that had been pretreated systemically with LPS. These effects were not due to neutropenia, as neutrophil counts had already returned to baseline 3 h before the last mice were sacrificed to examine local neutrophil emigration (Fig. 1GoA; please note intravascular neutrophils in Fig. 2GoJ). Importantly, L-selectin expression on animals treated systemically with LPS and controls did not differ significantly (Fig. 1GoC). Thus, L-selectin shedding does not account for the observed total inhibition of neutrophil emigration as part of the systemic inflammatory reaction.

Blood was taken at all time points throughout the experiments, and neutrophils were stimulated with LPS. Neutrophils of mice that had been injected systemically with LPS i.p. remained fully responsive toward the relatively low dose of LPS administered ex vivo (50 ng/ml, 37°C, 30 min). If anything, the degree of additional Mac-1 up-regulation or L-selectin shedding (data not shown) was more pronounced in neutrophils from mice that had received LPS i.p. than that seen in control animals (Fig. 3Go). Moreover, the degree of E-selectin and VCAM-1 up-regulation on endothelial cells of mice who had received LPS both systemically and locally was higher than in those animals who received LPS either only systemically or only locally (Table IGo; differences in ICAM-1 expression were difficult to interpret due to the high degree of ICAM-1 expression at baseline). When mice, systemically pretreated with LPS, were locally injected with IL-8 or TNF instead of LPS, neutrophil emigration was also inhibited (Table IIIGo). Thus, the inhibition of neutrophil emigration into areas of local inflammation, which is part of the systemic inflammatory reaction in animals preinjected with LPS i.p., is not due to tachyphylaxis of either murine endothelium or neutrophils toward LPS.



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  		FIGURE 3. Systemic exposure of neutrophils to LPS in vivo does not cause cellular desensitization toward restimulation by LPS ex vivo. Blood was taken from SV-129 mice before (white bars) and 170 min after the systemic application of LPS (10 µg LPS in 200 µl and 154 mM NaCl i.p.; black bars) and either left on ice or stimulated ex vivo by LPS (50 ng/ml, 30 min, 37°C), as indicated. Mac-1 (M1/70, rat anti-mouse mAb) expression was determined on peripheral neutrophils stained by indirect immunofluorescent technique (flow cytometry, FACScan).

 

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  		Table III. Inhibition of leukocyte emigration by systemically applied LPS toward locally applied LPS, TNF-{alpha}, and IL-81

 
The inhibitor of neutrophil emigration induced in the course of the systemic inflammatory reaction following the i.p. injection of LPS is not IL-8

When mice were injected systemically with TNF instead of LPS, leukocyte emigration toward the local inflammatory stimulus of LPS was also inhibited. Interestingly, anti-TNF mAb abrogated the inhibitory activity of systemically applied TNF at various concentrations, but failed to do so for systemically applied LPS (Table IVGo). These results imply that LPS, which does induce the production and secretion of TNF as part of the inflammatory reaction it causes, initiates the inhibition of leukocyte emigration not only by the TNF pathway. In addition, it can be assumed that TNF itself is not the putative inhibitor of leukocyte emigration.


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  		Table IV. Inhibition of leukocyte emigration by LPS and TNF1

 
It has been shown previously that the systemic pretreatment of rabbits with IL-8 (20 µg/kg, i.v.) may inhibit emigration of neutrophils from rabbit mesenteric venules induced by the local application of IL-8 into the soft tissues of the mesenterium. This inhibition was observed until about 50 min after the initial systemic administration of IL-8 without altering neutrophil L-selectin expression or leukocyte rolling (at plasma levels of IL-8 above 0.3 ng/ml) (23). To test whether IL-8 is the inhibitor of neutrophil emigration induced during the systemic inflammatory reaction due to the i.p. application of LPS, SV-129 mice were injected systemically with IL-8 (50 µg/kg or 1.5 µg, respectively, i.p.) instead of LPS, followed at 120 min by the local injection of LPS (0.1 µg) into the right rear footpad using systemically untreated animals as controls. The i.p. injection of IL-8 was repeated twice at 90 min and at 180 min to keep plasma IL-8 levels above 10 ng/ml. We could demonstrate that the systemic application of IL-8 in mice leads to slight increases in Mac-1 expression without causing a simultaneous decrease in L-selectin expression, as previously reported in rabbits (23). In our model, however, systemic pretreatment of mice with IL-8 (in doses from 0.625 up to 5 µg) before the injection of LPS into a rear footpad of SV-129 mice did not inhibit neutrophil emigration into the site of LPS-induced local inflammation at 250, 275, 300, 325, and 350 min after the initial injection of IL-8 (Fig. 4Go; Table VGo). In addition, pretreatment of mice with anti-IL-8 rabbit antiserum before the i.p. injections with LPS did not abrogate the inhibition of leukocyte emigration at sites of local inflammation (LPS, 0.1 µg, right rear footpad), while it inhibited the activity of IL-8 in the same mice in vivo (IL-8, 5 ng, left rear footpad; Table VIGo) and neutralized the chemotactic activity of IL-8 in an in vitro Boyden chamber system (data not shown).



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  		FIGURE 4. Inhibition of leukocyte emigration induced during the systemic inflammatory reaction is not due to IL-8. SV-129 mice were left untreated for controls or pretreated with IL-8 (50 µg/kg, i.p.). At 120 min, the right rear footpad was injected with LPS (0.1 µg), and the left rear footpad with carrier solution only. Blood was taken at various time points to monitor neutrophil counts and neutrophil expression of Mac-1 and L-selectin (not shown). The animals were sacrificed 130, 155, 180, and 230 min after the local injection of LPS. Wright-Giemsa-stained thin sections of methacrylat-embedded right rear feet are shown (A, x125; B, x783). Results of control animals were similar to those depicted in Figure 2Go and Table IGo.

 

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  		Table V. Emigration of PMN at sites of local inflammation after systemic stimulation by IL-8 (50 µg/kg)1

 

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  		Table VI. Anti-IL-8 mAb does not abrogate the inhibition of leukocyte emigration induced by LPS1

 
In preliminary transfer experiments, no inhibition of neutrophil emigration into sites of local inflammation could be demonstrated. Although a positive transfer experiment would have strongly suggested a soluble inhibitor of leukocyte emigration induced by the systemic application of LPS, our negative results do not argue against an inhibitor or even its existence in soluble form. As LPS administered i.p. was detectable at a level of 1 pg/ml up to 150 min after the initial injection, the transfer could only be undertaken 180 min after the initial i.p. injection of LPS into the primary mouse; if not, the effect of LPS itself was to be tested. A soluble plasma factor might long have come and gone by that time (incidentally, the plasma t1/2 of most cytokines is short). To circumvent this particular problem, we have also tried to transfer the inhibitory activity of mouse plasma (30 min after i.p. injection of LPS) into an in vitro model of HUVEC, but no inhibitory activity could be detected. Again, these negative results do not argue against the existence of a soluble factor in the plasma of systemically inflamed mice in vivo, as the HUVEC system is highly artificial with respect to the in vivo situation, and its applicability implies cross-species inhibitory activity induced by the i.p. injection of LPS in mice. Furthermore, the inhibitor might just as well be membrane bound, for example on endothelial cells, and never appear in plasma.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The in vitro activation of endothelium with LPS directly induces the adhesion of neutrophils, and subsequently significantly increases spontaneous transmigration of neutrophils through the endothelial monolayer (11, 14, 15). In striking contrast, neither neutrophil adhesion to vascular endothelium nor tissue emigration can be observed in vivo in SV-129 mice after systemic exposure to LPS. Despite activation of both neutrophils and endothelial cells, including the up-regulation of cell surface adhesion molecule Mac-1 on neutrophils, and E-selectin, ICAM-1, and VCAM-1 on endothelium, the systemic application of LPS did not induce a generalized tissue emigration of neutrophils. It is important to emphasize at this point the difference of genuine tissue emigration of neutrophils and early neutrophil capillary retention in lung and liver, which correlates with early neutropenia following exposure to LPS (24, 25) and which is not the subject of our study.

To our knowledge, systematic studies examining this fundamental difference between in vitro models and the pathophysiologic sequence of events in vivo do not exist. However, our results might have been suggested by autopsy findings of patients who die of endotoxemic shock in the course of septicemia. Activation of neutrophils and endothelium has been demonstrated in patients with septicemia (26, 27, 28, 29, 30, 31, 32), but a generalized tissue invasion of neutrophils cannot be demonstrated (18). The results of our studies emphasize for the investigator possibly unaware of the pathologic findings in sepsis the important difference between the results of neutrophil transmigration experiments in vitro and neutrophil tissue emigration in vivo. In this light, the increased expression of Mac-1 expression in tissue observed after the systemic application of TNF-{alpha} in a mouse model of inflammation may turn out to reflect only increased Mac-1 expression on macrophages, as it did in our own study (33). It remains the question whether the lack of neutrophil emigration in vivo both in the mouse model and in sepsis is due to an inhibitor that is only induced in vivo or whether the transmigration of neutrophils seen in vitro is an artifact and results from prestimulation or additional stimulation of endothelial cells grown under in vitro conditions on plastic.

In this context, it is of great importance that the preexposure of SV-129 mice to systemic LPS results in the inhibition of neutrophil emigration at sites of local inflammation induced by a second s.c. injection of LPS. Inhibition of neutrophil emigration can be demonstrated even at time points when neutrophil counts in peripheral blood have long returned to normal. In comparison with neutrophil activation by LPS in vitro, which leads to the almost entire shedding of neutrophil cell surface L-selectin, L-selectin cell surface expression following systemic exposure of neutrophils to LPS differs not significantly from that of control animals. Therefore, the inhibition of neutrophil emigration at sites of local inflammation following preexposure of SV-129 mice to LPS is not caused by neutropenia that is only transient, nor by the shedding of L-selectin. Although cell surface up-regulation of Mac-1 was demonstrated following the systemic administration of LPS reflecting neutrophil activation, the conformational changes of Mac-1 molecules on mouse neutrophils indicating the activation of the integrin molecule itself were, due to the lack of activation epitope-specific mAbs, not formally shown. However, even if the systemic application of LPS in vivo does not cause integrin activation (which it does in vitro when we reexpose mouse PMN prestimulated with LPS in vivo to LPS ex vivo), this still does not explain the inhibition of leukocyte emigration at local sites of inflammation, where the local action of inflammatory mediators suffices to cause cellular adhesion, activation, cellular integrin activation, and emigration of leukocytes into tissue.

It might now be argued that the inhibition of neutrophil emigration is caused by tachyphylaxis toward a second stimulus of LPS (34, 35). However, even after preexposure with systemic LPS in vivo, neutrophils remain fully responsive toward additional stimulation with LPS ex vivo. In addition, the local injection of LPS leads to further increases in the expression of adhesion molecules on endothelial vessels in the rear footpads, even though these increases do not result in neutrophil emigration into the surrounding tissue. Importantly, the systemic pretreatment of mice with LPS inhibited the emigration of neutrophils at sites of local inflammation induced not only by LPS itself, but also by TNF and IL-8. Thus, tachyphylaxis of either neutrophils or endothelial cells toward LPS seems very unlikely as an explanation of the observed total inhibition of neutrophil emigration at sites of local inflammation. In conclusion, in contrast to neutrophil and endothelial cell interactions in vitro, the systemic exposure toward LPS in vivo induces a factor(s) that inhibits neutrophil emigration in vivo, despite up-regulation of neutrophil and endothelial adhesion molecules.

In search for the inhibitor of neutrophil emigration, we first turned to TNF, which is produced and secreted as part of the generalized inflammatory reaction caused by the systemic application of LPS (36). The systemic treatment of mice with TNF, quite like LPS, causes a complete inhibition of leukocyte emigration. However, while anti-TNF mAb abrogated TNF-induced inhibition of leukocyte emigration, it had no effect upon the inhibition of leukocyte emigration induced by LPS. Therefore, first, TNF most probably is not the putative inhibitor of leukocyte emigration, and, second, the systemic application of LPS induces the inhibition of leukocyte emigration not only by the TNF pathway.

The systemic application of IL-8 previously has been shown to inhibit by approximately 50% neutrophil emigration induced by the local application of IL-8 independently from alterations in L-selectin expression or leukocyte rolling in a rabbit animal model (23). In our system, in which we found a complete rather than only a 50% inhibition of leukocyte emigration induced by the systemic application of LPS or TNF, IL-8, at plasma levels well above those that proved inhibitory in the rabbit model, did not interfere with neutrophil emigration into sites of local inflammation. In contrast to our experimental model, neutrophil emigration from rabbit mesenteric venules was not induced by LPS, which is thought to be the most potent agent capable of inducing neutrophil efflux (37), but by a single chemokine, IL-8 (23), with an observation period of only 50 min, which might explain the discrepancies in results. Furthermore, pretreatment of mice in our system with anti-IL-8 rabbit antiserum did not influence the inhibitory activity induced by systemically applied LPS, although it neutralized the effects of IL-8 in the same animals. We conclude that IL-8 is not the inhibitor of neutrophil emigration that is induced during the systemic inflammatory reaction in LPS-treated mice. In this context, it may be important to note that neutrophil emigration is greatly reduced and not increased in IL-8R knockout mice (38).

The putative inhibitor of neutrophil emigration is probably of great physiologic importance, because it prevents in vivo the generalized emigration of activated neutrophils into all kinds of tissue during a systemic inflammatory reaction, such as in sepsis. The excess expression of the inhibitor, however, may seriously imperil a patient’s defense in sepsis against local microbial invasion, as, for example, in a developing pneumonia. Experiments are in progress to further characterize and molecularly define this newly recognized inhibitor of neutrophil emigration.


   Acknowledgments
 
We thank Kathy Mayr for excellent technical assistance. Special thanks are due to Dr. D. Vestweber for providing mouse E-selectin-specific mAb 10E9.6.


   Footnotes
 
1 This work was supported by Swiss National Science Foundation Grant 31-39602-93. Back

2 Address correspondence and reprint requests to Dr. B. Schleiffenbaum, A-Hof-143, Division of Hematology, Department of Internal Medicine, University Hospital Zürich, Rämistrasse 100, CH-8091 Zürich, Switzerland. Back

3 Abbreviation used in this paper: PMN, polymorphonuclear. Back

Received for publication August 7, 1997. Accepted for publication May 18, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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