Hepatocyte growth factor stimulates neutrophil degranulation but not respiratory burst

Neutrophil function is regulated in part by cytokines with growth factor activities for different cell types. Hepatocyte growth factor (HGF) is a cytokine produced during injury to the liver and other organs. Neutrophils are numerous in such tissue injury sites and may be influenced by HGF. In the present study the effect of HGF on neutrophils was investigated. The data show that HGF at 1–10 ng/ml increased lysosomal enzyme release from both specific and azurophilic granules of cytochalasin-B treated neutrophils. The release of specific granule contents in response to N-formyl-methionyl-leucylphenylalanine was also increased by HGF. In contrast there were no significant effects of HGF on neutrophil respiratory burst, adherence or locomotion. It is concluded that HGF modulates neutrophil granule exocytosis.


Introduction
Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and is found in the sera of animals with liver injuries. It is now known that HGF is a pleiotropic factor (reviewed in References 2 and 3) with effects including promotion of epithelial cell growth, 2 inhibition of proliferation of several tumour cell lines, 4 enhancement of epithelial cell motility and identity with scatter factor and induction of tubule formation by epithelial cells. 2 The cDNA of HGF has been cloned and the HGF molecule has been characterized as a heterodimer consisting of a 65 kDa z chain and a 34 kDa/3 chain, structurally similar to plasmin. 3 HGF is produced by Kupffer cells in the liver, and also in other organs such as lung and kidney, not only in liver injury but also after renal damage (reviewed in References 2 and 3). The HGF receptor is the c-met proto-oncogene product, and is widely distributed. Neutrophil granulocytes are important mediators of both host defence against microbial attack and tissue damage by virtue of their armament of lysosomal granule enzymes and products of the respiratory burst. The function of mature neutrophils can be regulated by cytokines and growth factors such as interleukin-1 (IL-1), 7'8 tumour necrosis factor-alpha (TNF00, 7'9 and granulocyte macrophage colony-stimulating factor (GM-CSF) 1'11. HGF, like IL-1 and GM-CSF, can be produced by cells of the monocyte lineage. 12 '13 Neutrophil counts are likely to be elevated during the traumatic injuries which can lead to high HGF (C) 1993 Rapid Communications of Oxford ktd production. A form of HGF known as tumour cytotoxic factor derived from fibroblasts induces differentiation of HL-60 cells into the mature granulocyte phenotype. 14 GM-CSF, which promotes the differentiation of granulocyte precursors also enhances the function of mature granulocytes. 1'1' Therefore HGF was examined for its effects on neutrophil functions.

Materials and Methods
Hepatocyte growth factor: Human recombinant HGF was produced in transformed Chinese hamster ovary cells. 16  were added to the tubes immediately before placing them into the luminometer (Model 1250, LKB Wallac, Turku, Finland). Maximal rate of chemiluminescence was recorded as mV. Superoxide dismutase inhibitable superoxide production was measured as described previously after preincubation of neutrophils with HGF as above, and stimulation with or without FMLP.
Adherence of neutrophils: Neutrophil adherence to plasma coated plastic microtitre wells was measured as described 21 using a modified spectrophotometric assay. 22 Expression of results and statistics: Within each experiment, assays were conducted in at least triplicate, using neutrophils from a single donor. Each experiment was conducted three times or more with neutrophils from different donors. Results are presented as means (+_ S.E.M. or range) of replicate experiments. The significance of the data was tested using Wilcoxon's matched pairs signed ranks test.

Results
Effects of HGF on neutrophil granule exocytosis" HGF induced the degranulation of neutrophils. Preincubation with recombinant human HGF for 20 min increased the release of both specific and azurophilic granule components from cytochalasin-B treated neutrophils in a dose dependent manner (Fig. 1). HGF concentrations during preincubation of 1, 5 and 10 ng/ml enhanced the release of Vit B12 BP, a marker of neutrophil specific granules, to almost double the release from control neutrophils ( Table 1, Fig. 1). The release of/-glucuronidase (azurophilic granule marker) was also augmented by HGF at 5 and 10ng/ml (Table 1, Fig. 1). Although these increases were relatively small (granule release was less than 10% of the total cell content, even with HGF stimulus), they were statistically significant in a group of nine separate experiments performed with neutrophils from dierent donors, evaluated with Wilcoxon's matched pairs signed ranks test (Fig. 1). Neutrophils were preincubated with either HGF (10ng/ml) or diluent control for 20min before the addition of stimuli, b Neutrophils were further treated with various combinations of cytochalasin-B (2/g/ml final) or diluent for 10 min, followed by FMIP (5 x 10-6M final) or diluent, Assay values for control neutrophils (without HGF) are shown as mean percent of total cell content released for Vit B12 BP and -glucuronidase, maximal rate of light emission in mV for chemiluminescence, and change in optical density at 570 nm for adherence, The effect of HGF is expressed as a percentage of the control assay value (mean and range) for the specified number of experiments. potentiated by HGF at 5 ng/ml (Fig. 1). Other concentrations of HGF did not significantly enhance FMLP elicited Vit B12 BP release, nor was fl-glucuronidase release significantly increased after these conditions (Fig. 1, Table 1). HGF did not affect neutrophil granule enzyme release in response to PMA or TNF0 (results not shown).
LDH release from neutrophils was not detectable without FMLP stimulation and was always <7% with FMLP. LDH release was unaffected by HGF (results not shown), indicating that HGF did not impair cell viability or cytosol membrane integrity.
In experiments in which cytochalasin-B was omitted there was no significant effect on HGF on either Vit B12 BP or fl-glucuronidase release, nor was there any consistent or significant effect of HGF on FMLP elicited degranulation under these conditions ( Table 1).
Treatment of neutrophils with 100U/ml of TNF for 30 min is known to prime the cells for increased enzyme release, 7 and HGF was examined for its ability to modify this function. Incubation of neutrophils .with 10 ng/ml HGF immediately after TNF treatment had no consistent efl:ect on release of either Vit B2 BP or fl-glucuronidase, with or without additional FMLP stimulation (results not shown).
Eect of HGF on neutrophil respiratory burst: Lucigenin enhanced neutrophil chemiluminescence was used to measure the respiratory burst. Neutrophils were preincubated for 20 min with a range of concentrations of HGF, followed by 10 min with cytochalasin-B, before monitoring luminescence in response to FMLP or diluent. HGF at 1 and 10 ng/ml during preincubation increased basal chemiluminescence slightly ( alter their random migration under agarose, nor oxidase rather than increased respiratory burst their migration towards the chemotactic agent activity.

FMLP (results not shown).
It is unlikely that the increase in granule release seen with .HGF was due to contamination with Discussion bacterial endotoxin because there was no effect on respiratory burst which would be expected with It is reported here that recombinant human HGF lipopolysaccharide. 28 The recombinant HGF was induces the release of granule enzymes from prepared in transformed mammalian cells and the neutrophils. Secretion of both Vit B2 BP and stock solution (200ng/ml) did not contain any fl-glucuronidase, markers of the specific and measurable TNFfl, TNF or IFN-2 (i.e. <100 azurophilic granules, respectively, was enhanced up pg/ml) by enzyme-linked immunosorbent assay to 200%, suggesting that HGF is a complete performed in our laboratory. There was also no secretagogue for human neutrophils. The effect was effect of HGF on the other neutrophil functions that dose dependent and required only 10 min prewere tested. These included adherence to plasmaincubation. HGF shares this ability to induce the coated plastic and random or chemotactically exocytosis of both types of neutrophil granule with directed migration under agarose. Taken together other cytokines e.g. IL-1 v'8 and TNFfl. 23 The these results suggest that HGF is able to induce magnitude of the increase in enzyme release caused neutrophil granule release without affecting other by HGF was similar to that observed with TNF, 23 functions.
GM-CSF 1 and interferon gamma (IFN-2), 24 stimu-It is not known if neutrophils express receptors lation of neutrophils. The increased release of for HGF, but this receptor, known to be the c-met granule enzymes from HGF treated neutrophils was proto-oncogene product, is widely distributed on not due to HGF toxicity because viability (LDH many cell types. 6 The high affinity receptor on release) was not altered, hepatocytes has a Kd of 60-90 pM, corresponding Neutrophil specific granule release (Vitamin B12 to about 5-8 ng/ml. 2 This study and others which BP) in response to FMLP/cytochalasin-B was describe effects of HGF on neutrophils 2v and the increased by HGF at 10 ng/ml only. There was no myeloid cell line HL6014 suggest that neutrophils effect on /3-glucuronidase release under these might bear HGF receptors.
conditions, nor was there any effect of HGF on Other growth factors can modulate neutrophil TNF priming of neutrophil exocytosis. These activities. For example growth hormone and results suggest that HGF does not prime insulin-like growth factor-1 prime neutrophils to neutrophils for an increased response to subsequent secrete superoxide. 29 Platelet derived growth factor stimulation in contrast to other cytokines such as upregulates the 5-1ipoxygenase pathway in differ-TNFo, v'9 IFN-24'2s or GM-CSF. '11 Cytochalasin-B entiating neutrophils. Transforming growth was required in these experiments in order for HGF factor-beta induces precursor cells (CFU-GM) to to exert a measurable effect on basal enzyme release, differentiate preferentially into granulocytes. 31 Cytochalasin-B is a fungal metabolite which Other cytokines which influence cell growth and interferes with the microtubule contractile system differentiation such as the interleukins and colonyand phagocytosis. 26 It enhances granule release and stimulating factors, can activate neutrophil funcrespiratory burst in response to otherwise weak or tions including adhesion, migration inhibition, inactive soluble or particulate agonists. 26 The antibody dependent cytotoxicity, triggering of release ofneutrophil lysosomal enzymes in response respiratory burst and degranulation, priming for to IL-1 and TNFo also required cytochalasin-B, v'8 increased responses to stimuli, microbicidal action The present data show that neutrophil respir-and cartilage damage. 7-11'21-25 atory burst activity was not significantly altered by Since HGF affects differentiation of granulotreatment with 0.1-100 ng/ml HGF compared to cytes 14 these findings support the hypothesis that diluent. These results apparently contrast with a factors which regulate myeloid growth and recent report that HGF primes neutrophils for differentiation also modulate the function of the increased luminol enhanced chemiluminescence. 2v mature cell. HGF is produced in several conditions [1][2][3] We measured lucigenin enhanced chemilumin-such as hepatic failure and renal injury. escence which is a measure of superoxide release, 28 Neutrophils are likely to encounter high concentrathe primary oxygen radical produced by neu-tions of HGF at such times. For example serum trophils. Since chemiluminescence enhanced by HGF concentrations of >1 ng/ml have been luminol measures formation of other down-stream reported in 82% of patients with fulminant hepatic reactive oxygen metabolites such as hypochlorous failure, rising to 5-10 ng/ml immediately after acid which is dependent on myeloperoxidase release plasma exchange. 32 HGF concentrations in the from the specific granules, 28 the results of Jiang et microenvironment around the HGF-producing al. 2v may reflect an increased release of myeloper-cells at injury sites are probably even higher.
Neutrophil precursor cells have also been reported to produce HGF. 12 It has already been shown that HGF is a pleiotropic factor with endocrine, paracrine and autocrine effects on a number of dierent cell types. [1][2][3] In this paper it is reported that HGF at physiological concentrations induces lysosomal enzyme release from neutrophils. Since neutrophils are so numerous and have a half-life in the circulation of only about 7 h, their combined enzyme release in response to HGF could be significant. Other cytokines, such as TNFfl, GM-CSF, TNFo and IFN-2, which increase neutrophil azurophilic granule release can also augment neutrophil mediated cartilage damage in vitro. 21'23 Such host tissue damage is in large part due to lysosomal enzymes such as elastase. 33 HGF is likely to be one of many growth factors and cytokines which contribute to the complex regulation of immune cell functions.