Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal−) and bound (D-gal+) fractions, with MNCF being found in the D-gal+ fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal− fraction. Furthermore, the incubation of the D-gal− fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+ activity. Overall, these results suggest that the D-gal− inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migration in vivo by the D-gal+ fraction.


Introduction
Several studies have established that Gramnegative bacteraemia or circulating endotoxin decreases the ability of neutrophils to migrate into the inflammatory sites, a phenomenon which may play an important role in the evolution of sepsis. -It has been demonstrated that the impairment of neutrophil migration is mediated by inhibitory factors including tumour necrosis factor-cz (TNF-cz) 4 and interleukin 8  . 5 Previous work from our laboratory demonstrated that the intravenous administration of LPS-stimulated macrophage supernatants also inhibits neutrophil migration to the inflammatory site following, exposure to various stimuli. When the supernatants were submitted to gel filtration on Sephacryl S-300, the in vivo neutrophil chemotactic inhibitory activity was detected mainly in a fraction which eluted in a volume corresponding to high molecular weight proteins (240-550 kDa). This fraction also inhibited the cell-dependent oedema induced by carrageenin 116 Mediators of Inflammation Vol 5 1996 or by ovalbumin in previously immunized rats. The cell-independent oedema induced by dextran was not affected by the S-300 active fraction. 7 This fraction was also able to inhibit the increase in neutrophil and eosinophil numbers in the bronchoalveolar lavage fluid of sensitized guinea-pigs as well as the ovalbumin-induced bronchoconstriction in these animals. 8 Recently, the purification of a 54kDa acidic protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. 9 The in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines, such as TNF-cz and IL-8. MNCF induces neutrophil migration through a carbohydrate-recognizing property.
Since NRIF and MNCF are obtained under the same in vitro conditions, i.e. in the supernatants of LPS-stimulated macrophages, MNCF activity is also detected in the S-300 active fraction, 7 we have investigated whether MNCF account, at least in part, for the NRIF activity. For this, we have (C) 1996 Rapid Science Publishers purified macrophage supernatants using a simple the D-gal + fraction was further chromatographed two-step process involving adsorption to a D-on a Superdex 75 column. For this, the fraction galactose column followed by gel filtration on was concentrated to 0.2ml and applied to a Superdex 75. We have demonstrated that TNF-a Superdex 75 HR 10/30 column (Pharmacia LKB and IL-8 mediate the activity of the D-galfraction Biotechnology, Uppsala, Sweden) previously and that MNCF is responsible for the in vivo equilibrated with PBS and calibrated with known neutrophil recruitment inhibitory activity of the molecular weight markers. Fractions of 0.5ml D-gal + fraction, were collected and tested .in vivo or in vitro.

Materials and Methods
In vivo neutrophil migration assay..
Animals: Male Wistar rats weighing 180 to 200 g Inhibition assay. Crude supernatant, D-gal + Dand housed in a temperature-controlled room galor Superdex 75 chromatographic fractions received water and food ad libitum. The rats were injected intravenously (i.v.) into the penial were used as the source of peritoneal macro-venous sinus of rats. Each animal received maphages as well as for the in vivo assays of neu-terial equivalent to that released by a 6 x 10 trophil migration inhibition, macrophages in 0.2ml. Thirty min later, the animals received an intraperitoneal (i.p.) injec-Production of macrophage supernatants: The tion of carrageenin (300 l.tg/ml) (Marine Colmethod for obtaining crude macrophage superloids, Inc., USA) while the controls (C) received natants containing the inhibitory activity has been PBS. After 4 h, the animals were sacrificed and described in detail. 4 Briefly, rat macrophages their peritoneal cavities washed with 10ml of were harvested from peritoneal cavities elicited 4 PBS containing heparin (5 IU/ml). Total and difdays earlier with 10 ml of 3% thioglycollate (w/v) ferential cell counts were performed as described and incubated in tissue culture dishes for i h at elsewhere, and the results were expressed as 37C, in an atmosphere of air containing 5% the percentage (%)of inhibition. CO2. The adherent monolayers were washed three times with sodium phosphate-buffered Effect of anti-TNF< and anti-IL-8 antisera on saline (PBS, pH 7.4) and incubated with LPS (5 the inhibition of neutrophil migration caused by btg/ml of RPMI) for 30min at 37C. The cells + the D-galand D-gal fraction. The D-galor D-+ 6 were again washed three times with PBS fol-gal fraction (obtained from 6 x 10 cells)was lowed by a final incubation with LPS-free incubated with sheep antisera (20 l.tl/200 I.tl of medium, for 90 min at 37C. The cell-free incu-sample) against murine recombinant TNF-a bation medium was centrifuged (2000 x g for (mrTNF-a) and against human recombinant IL-8 10min at 25C) and subsequently ultrafiltered (hrlL-8) in a 5% CO2 atmosphere at 37C, for through a YM-10 membrane (Amicon Corp., Lex-30min. As a control, the fractions were also ington, MA, USA) against sterile, deionized water incubated with normal serum. Subsequently, at 4C. The supernatants were then concentrated each fraction was administered i.v., 30min to 5 ml and filtered through a 0.22 l.tm membrane before the i.p. injection of carrageenin (300 t.tg). (Millipore, Bedford, MA, USA). This preparation After 4h, total and differential cell counts were was designated as the crude supernatant, and performed and the results were expressed as was either used for in vivo or in vitro experi-the number of neutrophils x 10/mi of lavage ments, or was chromatographed on an agarose/ fluid. D-galactose column.
TNF activity assay. The TNF content in the Chromatographic procedures for the fractiona-crude supernatant and in the chromatographic tion of crude supernatant: Partial purification of fractions was measured using a highly TNF-senthe crude supernatant was performed as descrisitive cell line, WEHI 164 clone 13, as described bed by Dias-Baruffi et al. 9 Briefly, crude super-elsewhere. 2  were added to each well and the plates incubated for an additional 4h. Subsequently, 100 l, tl of isopropanol containing 0.04N HCl were added to each well. Fifteen min later, the degree of cell lysis was quantitated spectrophotometrically (570nm) by using an enzyme-linked immunoassay analyser (Multiskan MCC/340 MKII, Flow Laboratories). Because of the unavailability of rat TNF-a, standard curves were prepared with mrTNF-a (Genentech Inc.. When the D-galfraction was co-incubated with anti-TNF-a plus anti-IL-8 antisera, its inhibitory activity was partially prevented. In contrast, the same treatment did not alter the inhibitory effect of the D-gal + fraction on neutrophil migration (Fig. I(B)).
Eect of Superdex 75 fractions on the inhibition of neutrophil migration: The D-gal + preparation was chromatographed on a Superdex 75 column and the ability of some of the fractions to inhibit neutrophil migration in vivo was tested. Figure 2 shows that the inhibitory activity present in the crude supernatant was detected in the fraction eluted in a volume of 10 ml, corresponding to an apparent molecular weight of 54kDa, as shown by the chromatographic profile. In previous work this fraction was also demonstrated to contain neutrophil chemotactic activity. Electrophoretic analysis of the D-galfraction showed was recovered mainly in the D-galfraction. In the D-gal + fraction, only small amounts of TNF were found (7.69/0 of cells killed), and this decreased to almost zero after chromatography on Superdex 75. Antibodies raised against mrTNF-a abolished the cytotoxicity of mrTNF-a (data not shown) and strongly reduced the activities of the crude supernatant and the D-galfraction, thus confirming that the cytotoxic activity observed was due to TNF (Table 1).

Discussion
In the present paper, we have confirmed previous results showing that the intravenous administration of LPS-stimulated macrophage supernatants inhibits the neutrophil migration induced by 6 carrageenin, LPS or Pseudomonas aeruginosa. An affinity chromatographic procedure using a D-galactose affinity column showed that the inhibitory activity was recovered in both the bound and unbound fractions.
The inhibitory effect presented by the D-galfraction was due, at least in part, to the presence of TNF-a, since a significant amount of this cytokine was found in this fraction. In support of this, incubation of the D-galfraction with anti-TNF-a plus anti-IL-8 antisera partially prevented the inhibitory action. In this context, TNF-a has been reported to inhibit neutrophil migration in different animal models. 4'13 The anti-IL-8 antiserum was employed because we have previously detected IL-8 in samples of crude LPS-stimulated macrophage supernatants (data not shown), and this cytokine is also known to inhibit neutrophil migration. 5'13'4 In contrast, the D-gal + effect was not due to contamination with TNF or even to the presence of IL-8, since no TNF was detected in the Superdex 75 fraction, nor did the anti-TNF plus anti-IL-8 antisera have any effect on the ability of the fraction to inhibit neutrophil migration.
Following further gel infiltration of the D-gal + fraction on Superdex 75, the inhibitory activity was recovered in a single fraction equivalent to a molecular weight of 54 kDa. Recently, we described the purification of a 54kDa acidic protein, identified as MNCF. 9 This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. Interestingly, in the present study, the neutrophil inhibitory activity was located in the same chromatographic fraction as that in which MNCF was previously detected, suggesting that the same protein accounts for both chemotactic (MNCF) and inhibitory (NRIF) activities.
These results are consistent with literature data which have already demonstrated that the proinflammatory chemotactic cytokines TNF-a and IL-8, when administered i.v., are able to inhibit the neutrophil recruitment to the inflammatory site. 4,5,3,4 Our results suggest that the inhibitory effect of the D-galfraction is mediated by TNF-a and IL-8. Moreover, in relation to the D-gal + fraction, the chemotactic and inhibitory activities on neutrophil migration are due to the same protein, already identified as a 54 kDa acidic protein. The mechanisms by which TNF-a, IL-8 and MNCF inhibit neutrophil migration are currently under investigation.