Tumour necrosis factor-alpha and interleukin-8 inhibit neutrophil migration in vitro and in vivo

Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-α) and/or interleukin-8 (rIL-8), but not with either transforming growth factor-beta, interleukin-6 or interferon-gamma, rendered these cells less responsive to FMLP, in microchemotaxis assays. This inhibitory effect was dose dependent and more powerful when neutrophils were pretreated with a mixture of both cytokines. Intravenous injection of human rIL-8 (hrIL-8) and/or murine rTNF-α (mrTNF-α) also significantly reduced in vivo neutrophil migration into peritoneal cavities of rats stimulated with carrageenan. These data suggest that the defect in neutrophil migration during septicaemia or endotoxaemia may be the result of the continuous release of IL-8 and TNF-α into the circulation. Thus, either the selective control or blockade of releasing of these cytokines as well as of its effects on neutrophils may be clinically useful in reestablishing the cell defence mechanisms.


Introduction
Several studies have established the fact that Gram-negative bacteraemia or circulating endotoxin decreases the ability of neutrophils (PMN) to migrate to inflammation sites. [1][2][3] This effect may play an important role in the evolution of septicaemia. 1'4 Neutrophil inhibitory factors have been found in many other illnesses, such as AIDS, Hodgkin's disease, 6 diabetes mellitus and lepromatous leprosy. The source of these factors has not yet been satisfactorily demonstrated. It has been shown that macrophages incubated with Staphylococcus aureus release a factor(s) which inhibits neutrophil migration in vitro. 9J Recently it has been shown that the supernatant of rat macrophages pretreated with lipopolysaccharide (LPS) administered intravenously (i.v.) suppresses the recruitment of neutrophils to the peritoneal exudate induced by various inflammatory stimuli, thus mimicking the effects of LPS. The factor(s) present in this supernatant was named NRIF (neutrophil recruitment inhibitory factor). 1 Tumour necrosis factor-alpha (TNF-o 0 and interleukin 8 (IL-8) exert a wide spectrum of activities in inflammatory reaction. They are potent stimulators of several neutrophil functions, including chemotaxis, respiratory burst, degranulation and aggregation. [2][3][4][5][6][7][8][9] Besides these pro-inflammatory effects, it was recently demonstrated that both cytokines have anti-inflammatory properties. 13'2-24 TNF-o blocks in vitro chemotaxis of human and rabbit neutrophils induced by several stimuli, such (C) 1992 Rapid Communications of Oxford Ltd as f-methionyl, 1-1eucyl phenylalanine (FMLP) Cha and LTB420-22'24 and IL-8 inhibits neutrophil adhesion to cytokine activated endothelial monolayers resulting in the protection of these cells from neutrophil mediated damage in vitro. 2 It was also shown that the intravenous injection of TNF inhibited Cha induced neutrophil emigration into skin of mice, 24 and that systemic administration of IL-8 caused a similar effect in the skin of rabbits challenged with FMLP, IL-1, C5a or LTB4 .2s In the present investigation the effect of TNFand IL-8, alone or in combination, on neutrophil migration has been investigated in vitro. The effect of transforming growth factor-fl, interleukin-lfl, interleukin-6 and interferon-gamma were also tested. Since TNF-0 and IL-8 inhibited neutrophil chemotaxis induced by FMLP, the effect of intravenous administration of these cytokines on carrageenan induced neutrophil recruitment in peritoneal cavities of rats was also tested.

Materials and Methods
Animals: Adult male Sprague-Dawley rats weighing 150-180g were obtained from Charles River Breeding Laboratories (Wilmington, MA).
Intravenous administration of mrTNF-0, hrlL-8 or a mixture of both reduced neutrophil migration induced by an i.p. injection of carrageenin. Similar to the in vitro results, mrTNFwas more potent than hrlL-8 in relation to the inhibition of neutrophil migration into the peritoneal cavities of rats (Fig. 4). used. TNF inhibition of neutrophil migration has consistently been seen with the agarose method, 2'26-28 while the chemotaxis chamber methods have yielded contradictory conclusions. 12'20'22'29 In the present experiments, hrTNF-0 and hrlL-8 inhibited neutrophil migration in vitro in a dose dependent manner. While TNF-0 was more potent than IL-8, the combination of these cytokines had an additive inhibitory eect. The high doses of hrlL-8 necessary to inhibit human PMN chemotaxis suggests that the cytokine is acting by crossreacting with a receptor for another ligand, or through cross-desensitization.
It is known that a high concentration of a chemotactic factor placed in the upper compartment of a chemotactic chember can inhibit neutrophil chemotaxis. 3 However, the inhibitory effect described in this paper was not due to the presence of residual TNFand IL-8 in the top chamber, since exposure of the neutrophils to mrTNF-, hrIL-8 or both, followed by cell washing, had the same in vitro inhibitory effect.
Intravenous injection of a single dose (2/2g/animal) of hrlL-8 and/or mrTNF-cx before the intraperitoneal administration of carrageenan caused significant reduction of neutrophil accumulation on rat peritoneal cavities. It has recently been reported that systemic administration of TNF 24'31 or It-8 2s'32'33 induces only a transient neutropenia which is followed by neutrophilia. Thus, the inhibitory effects observed in the present investigation probably are not due to neutropenia. It has been shown that IL-8 blocks neutrophil adhesion to activated endothelial monolayers 23 as well as inducing the loss of lectin adhesion molecule I (LECAM-1) from the unstimulated neutrophil surface, thereby reducing neutrophil adhesion to vascular endothelium under conditions of flow. 34 These findings may explain the IL-8 induced inhibitory effect observed here. The low efficiency of IL-8 in comparison to TNF-a in rat studies perhaps may be due to a weak recognition of hrIL-8 by rat PMN. In this regard, recent studies have shown that IL-8 may have some species specificity and then its chemotactic potency can vary according to the PMN source.
Previous studies have suggested that TNF-cz inhibits neutrophil migration in vitro by increasing the expression of the CD11b, thus resulting in enhancement of neutrophil adhesion and suppression of migration. 2 The authors showed that the monoclonal antibody 60.1, which is directed against an epitope on the alpha chain of the CD11b/CD18 complex, 36 completely blocked the inhibition of neutrophil migration induced by hrTNF-. This monoclonal antibody also completely reversed the hrTNFinduced hyperadherence to gelatin-coated wells. 2 The TNF-cx induced inhibition of neutrophil emigration into peritoneal cavities described in this paper, may also be due to the hyperadherence of neutrophils in the circulation which impairs the migration of these cells into extravascular space.
TNFhas been implicated as an important mediator of Gram-negative sepsis, which involves extensive polymorphonuclear mediated vascular and tissue damage. 37 TNFinjection into experimental animal produces a virtually identical 'septic shock syndrome' to endotoxin administration. 38 The animals passively immunized against TNFsurvive an otherwise lethal dose of endotoxin. 39 It has recently been shown that IL-8 also appears in the circulation of primates during septic shock, sublethal endotoxaemia and after IL-1 administration. 4 Thus, several events observed in the endotoxaemia, such as impairment of neutrophil migration, may be induced by release of IL-8 and TNF-0. Recently Cunha and coworkers, 11 have suggested that failure of neutrophils to migrat.e during septicaemia or endotoxaemia may be the result of a continuous release of a recruitment inhibitory factor (NRIF). Since NRIF is present in the supernatant of LPS pretreated macrophages, and since LPS also stimulates macrophages release IL-8 and TNF, probably part of inhibitory effect of the macrophage crude supernatants is due to the presence of TNF-cx and IL-8 in those samples.
In conclusion, the present findings suggest that TNFand IL-8 may be responsible for the inhibition of neutrophil migration found in septicaemia or endotoxaemia. Selective control of TNFand IL-8 or blockade of their effects on neutrophil migration may be clinically useful in reestablishing the cell defence mechanisms impaired in inflammatory diseases.