HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berkova, N. Articles by Naccache, P. H. Search for Related Content PubMed PubMed Citation Articles by Berkova, N. Articles by Naccache, P. H.

TNF-Induced Haptoglobin Release from Human Neutrophils: Pivotal Role of the TNF p55 Receptor¹

Nadia Berkova^2,*,, Caroline Gilbert, Serge Goupil^*, Ju Yan, Vyatcheslav Korobko and Paul H. Naccache

^* Laboratoire d’endocrinologie de la reproduction and Unité de recherche en genétique humaine et moléculaire, Centre de recherche de St-Françoise d’Assise, Centre Hospitalier Universitaire de Québec, Pavillon Saint-Françoise d’Assise, Québec, Canada; and Centre de recherche en rhumatologie et immunologie, Centre Hospitalier Universitaire de Québec, Pavillon Centre Hospitalier de l’Université Laval, and Department of Medicine, Laval University, Sainte-Foy, Québec, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Haptoglobin (Hp), TNF-, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF- on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF- induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF- was not due to nonspecific lysis of the cells. TNF- is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF- mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF- for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF--induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF- may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
To isolate and inactivate infectious organisms and to counteract tissue damage caused by injury, trauma, or infection, a complex set of early reactions known as the acute phase response (APR)³ is put into action by the host. Tissue macrophages and blood monocytes are the cells most commonly associated with initiating the cascade of events during the APR (1). Activated macrophages release a range of mediators, of which TNF- and IL-1 are critical for the progression of APR (2). These early pleiotropic cytokines interact with a wide variety of cells and induce the release of a secondary wave of cytokines that initiate the cellular reactions and the cytokine cascades of APR (3). Neutrophils are among the first cells to migrate to inflammatory sites, where they perform host defense functions including the phagocytosis of infectious organisms, the release of proteolytic enzymes, the generation of oxidized intermediates, and the synthesis and secretion of their own chemokines and cytokines such as IL-8, macrophage inflammatory protein-1, TNF-, IL-1, IL-1 receptor antagonist (4), and vascular endothelial growth factor (5).

The spectrum of systemic reactions to inflammation, the generation of the febrile response under hypothalamic control, the alterations in the level of essential metabolites and the regulation of genes in the liver, are closely associated with acute inflammation (6). The acute phase plasma proteins are generated by hepatocytes in response to tissue injury, infection, inflammation, or tumor growth (7). Haptoglobin (Hp), one of the acute phase reactant proteins shown to be involved in immune regulation (8), is a tetrameric glycoprotein consisting of two - and two ß-chains (9). The polymorphism of Hp is related to the heterogeneity of the -chain (15–20 kDa), whereas the glycosylated ß subunit is common to the different types of Hp. There are three major phenotypes of Hp: Hp-1 with the ¹-chain, Hp-2 with the ²-chain, and Hp2-1 with an ¹- and an ²-chains. The ²-chain differs from the ¹-chain in having an internal duplication of a large segment of the sequence.

In addition to a well-known hemoglobin-binding property, many other functions have been ascribed to Hp. It has been shown to inhibit cathepsin B activity and PG synthesis (9, 10). Hp has also been identified as one of the serum angiogenic factors required for the proliferation and differentiation of endothelial cells in the formation of new blood vessels (11). Furthermore, a Hp-like glycoprotein is expressed by endometrial cells in the very early stage of pregnancy in rabbits (12). It is possible that it functions as an antimicrobial factor or as a modulator of inflammatory/immune events occurring during the implantation process. Finally, we found decreased levels of autoantibodies to Hp-like protein in the serum of infertile patients (13).

Specific binding of Hp to neutrophils leading to an inhibition of respiratory burst activity has been reported. (14). Hp was also identified as an alternative ligand for the CD11b/CD18 integrin on neutrophils and monocytes (15). Moreover, it was shown that exogeneous Hp is taken up and stored in monocytes and neutrophils within a cytoplasmic granular compartment and is secreted by these cells during the phagocytosis of Candida albicans (16).

The present study was designed to investigate the effect of TNF- on the release of Hp from neutrophils. TNF- is a multifunctional cytokine that interacts with different cell types through two types of receptor with molecular masses of 55 kDa (p55) and 75 kDa (p75), respectively (17). These receptors may mediate cellular responses independently of each other (18). We have found that TNF- induces the release of Hp from human neutrophils and that administration of TNF mutants binding specifically either to p55 or to p75 TNFR showed that the release of Hp is predominantly mediated by the p55 receptor. The release of Hp from neutrophils induced by TNF- was dramatically decreased by erbstatin, a tyrosine kinase inhibitor.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

HBSS was purchased from Life Technologies (Burlington, Ontario, Canada). Ficoll-Paque was obtained from Pharmacia (Dorval, Québec, Canada). Human Hp was purchased from Sigma (St. Louis, MO). Recombinant human TNF- protein (1.3 x 10⁶ U/mg) was produced in the Institute of Bioorganic Chemistry, Laboratory of Gene Chemistry (Moscow, Russia) (19). The TNF- solution tested negative for pyrogenic activity.

The two recombinant human TNF mutants used in this study were generated by site directed mutagenesis and were kindly provided by Dr. Loetscher (Hoffman-La Roche, Basil, Switzerland) (20). The mutant TNF, which recognizes the TNFR p55 (TNFR-p55-specific mutant) is derived from the wild type of TNF- by two point mutations: Arg³² replaced by Trp and Ser⁸⁶ by Thr. The TNFR-p55-specific Trp³² Thr⁸⁶ mutant binds to TNFR-p55 with similar affinity as wild-type TNF- but shows no binding activity for TNFR-p75. The TNFR-p75-specific mutant was generated by replacing Asp¹⁴³ by Asn and Ala¹⁴⁵ by Arg. The TNFR-p75-specific Asp¹⁴³ Arg¹⁴⁵ mutant shows a 10-fold lower binding affinity for TNFR-p75 than wild-type TNF- and shows no binding activity for TNFR-p55. All products used for SDS-PAGE were purchased from Bio-Rad Laboratories (Mississauga, Ontario, Canada).

Neutrophil preparation

Whole blood from healthy adult volunteers was taken into heparinized tubes. Neutrophils were purified as we described previously (21). Briefly, the blood was centrifuged for 10 min at 1000 rpm; the platelet-rich plasma was removed, and the cell pellets resuspended in 2% dextran for sedimentation for 30 min. Afterward, neutrophils were purified by centrifugation over Ficoll-Paque cushions according to the manufacturer’s instructions. The remaining erythrocytes were removed by hypotonic lysis. The washed cells were then suspended in HBSS at a final concentration of 40 x 10⁶ cells/ml. The percentage of neutrophils in the cell preparations was >98% and cell viability exceeded 98% as determined by trypan blue exclusion and ethidium bromide penetration (22). Monocyte contamination, as judged by microscopic examination, was <0.2%. The procedure was conducted in sterile conditions at room temperature.

Cell stimulation

To demonstrate Hp release, 4 x 10⁶ cells in 100 µl of HBSS were stimulated with wild-type human TNF- at concentrations ranging from 0.13 ng/ml to 13 ng/ml for the prescribed periods of time at 37°C. As a control, an equivalent volume of the buffer solution, HBSS without TNF-, was added to the neutrophil suspension. In some experiments, the widely used neutrophil agonist fMLP (10^-7 M) was added to the suspension of neutrophils. In the experiments designed to investigate the role of TNF receptors in TNF--induced Hp release, either the Asp¹⁷³ Arg¹⁴⁵ mutant or the Trp³² Thr⁸⁶ mutant was added to neutrophil suspensions at the same concentrations as the wild-type TNF-. In some experiments, neutrophils (5 x 10⁶ cells/ml) were preincubated with 10 µg/ml of the tyrosine kinase inhibitor erbstatin (Sigma) for 30 and 60 min before stimulation with TNF- for 30 min. These conditions had previously been found to be optimal for the inhibition of the stimulation of tyrosine phosphorylation induced by TNF- in human neutrophils (23). Cell viability, after treatment with concentrations of TNF- up to 13 ng/ml at times ranging from 0 to 60 min, was measured by the trypan blue exclusion and ethidium bromide penetration tests (22).

Immunoblot analysis

The release of Hp by TNF--stimulated neutrophils was detected by Western blot analysis. Neutrophil suspensions (0.1 ml of 40 x 10⁶ cells/ml) were incubated with TNF- or HBSS for the indicated periods of time at 37°C. The suspensions were then centrifuged at 12,000 rpm for <10 s. The supernatants on the one hand and the cell pellets (resuspended in 100 µl of HBSS) on the other hand were transferred to microtubes containing an equal volume of 2x Laemmli’s sample buffer (62.5 mM Tris-HCl, pH 6.8, 8.4% SDS, 5% 2-ME, 8.5% glycerol, 2.5 mM orthovanadate, 10 mM paranitrophenylphosphate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.025% bromophenol blue) preheated to 100°C as previously described (21). The samples were kept at 100°C for 7 min before loading 25 µl of each solution onto 15% SDS-polyacrylamide gels according to the method of Laemmli et al. (24) in a Bio-Rad electrophoresis system (MiniProtean II; Bio-Rad Laboratories). Purchased human Hp standard (2 µg/ml) was processed in the same way.

After electrophoresis, the separated proteins were transferred electrophoretically at 4°C for 2 h at 250 mA onto 0.22-µm nitrocellulose membranes using a Mini Trans-Blot cell (Bio-Rad Laboratories). The nitrocellulose membranes were incubated in a blocking solution containing 10% skimmed milk in 0.1 M Tris buffer, 0.9% NaCl, 0.05% Tween 20, pH 7.2, and 3% normal goat serum (Sigma) overnight at 4°C. After washing, the membranes were incubated with rabbit anti-human Hp Ab (1:200) (Sigma) for 3 h, followed by an incubation for 1 h with goat anti-rabbit Ab (1:1000) coupled with peroxidase (Sigma). The membranes were then thoroughly washed and incubated with enhanced chemiluminescence (ECL) reagents (Amersham, Little Chalfont, U.K.) for 1 min, air dried, and wrapped in a plastic bag as described previously (21). Kodak X-OMATAR films (Eastman Kodak, Rochester, NY) were placed on the membranes and exposed for 20–60 s.

In some experiments, the rabbit anti-human Hp Ab were pretreated with excess Hp (40 µg/ml). The remaining steps were performed as described above.

Immunofluorescence

After preincubation of neutrophils (40 x 10⁶/ml) with 13 ng/ml of TNF- or HBSS for 1 h, 10 µl of the cell suspensions were placed on glass slides and the cells were fixed in a freshly made solution containing 2 volumes of formaldehyde, 19 volumes of acetone, and 19 volumes of MeOH for 20 min at -20°C (25). Because the fixation technique may influence the intracellular pattern of neutrophils (26), an immunofluorescence test on paraformaldehyde-fixed neutrophils was performed as described by Sternberger (27). Briefly, 10 x 10⁷ cells/ml were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in PBS, pH 7.4, for 10 min at 4°C. The slides were then incubated in 1% BSA, followed by a solution of 10% normal goat serum (Sigma). After washing, rabbit anti-human Hp IgG at a dilution of 1:100 was applied as primary Ab for 2 h at room temperature, followed by a biotinylated goat anti-rabbit IgG Ab at a dilution of 1:200 (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h. The slides were viewed with a fluorescence microscope after incubation with FITC-conjugated streptavidin 1:150 (Life Technologies) and staining of the nuclei with 20 µl of propidium iodide solution (25 µg/ml).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of TNF- on Hp release by neutrophils

The presence of Hp in the supernatant of neutrophil cultures stimulated by TNF- was examined by Western blot analysis. The rabbit anti-Hp Ab revealed several bands in the human Hp standard, corresponding to the ß-, ²-, and ¹-chains (Fig. 1, lane 1). Very low levels of Hp were detected in the supernatants of neutrophil control culture exposed to the buffer solution HBSS alone (Fig. 1, lane 2). A release of Hp from neutrophils was detectable at concentrations of TNF- as low as 0.13 ng/ml. At TNF- concentrations between 0.13 ng/ml and 13 ng/ml, three bands with molecular masses of 42 kDa, 20 kDa, and 16 kDa were detected in the supernatants in a concentration-dependent manner (Fig. 1, lanes 3–5). The presence of the three Hp bands indicates that the blood was received from donors of the Hp2-1 phenotype. Neutralization of anti-Hp Ab by preincubation with excess Hp (40 µg/ml) abolished the signal, confirming the specificity of the anti-Hp Ab (Fig. 1, lane 6). Cell viability tested by exclusion of trypan blue and ethidium bromide (22) was >98% under all conditions.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Concentration-dependent release of Hp from human neutrophils induced by TNF-. A constant volume of supernatant from TNF- stimulated or control human neutrophils (40 x 106/ml) and Hp standards were run on 15% SDS-PAGE as described in Materials and Methods. Neutrophils were stimulated with TNF- at the indicated concentrations. The separated proteins were transferred onto nitrocellulose membrane, blotted with rabbit anti-human Hp Ab (1:200), and incubated with a solution of peroxidase-labeled goat anti-rabbit Ab (1:1000). Bands were visualized by ECL. The specificity of the anti-Hp Ab was established by preincubation of the anti-Hp Ab with excess Hp for 2 h at room temperature. The arrows point to the ß-, 2-, and 1-chains of Hp. Molecular weight standards (10-3) are indicated on the left. This figure represents the results from seven experiments, Lanes 1–7, numbered from the left.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Kinetic of Hp release from human neutrophils treated with TNF-. Supernatants (lower panel) or extracts (upper panel) of human neutrophils, incubated with 13 ng/ml TNF- or HBSS for the indicated periods of time, were electrophoretically separated on 15% SDS-PAGE as described in Materials and Methods. The separated proteins were transferred onto nitrocellulose membrane, blotted with the rabbit anti-human Hp Ab (1:200), then incubated in a solution of peroxidase-labeled goat anti-rabbit Ab (1:1000). Bands were visualized by ECL. The arrows point to ß-, 2-, and 1-chains of Hp. This figure represents the results from five experiments.

View larger version (36K): [in this window] [in a new window]   FIGURE 3. Kinetic of Hp release from human neutrophils treated with f-Met-Leu-Phe. Supernatants (lower panel) or extracts (upper panel) of human neutrophils, incubated with 10-7M of f-Met-Leu-Phe or HBSS for increasing periods of time (20 to 60 min) were electrophoretically separated on 15% SDS-PAGE as described in Materials and Methods. The separated proteins were transferred onto nitrocellulose membrane, blotted with the rabbit anti-human Hp Ab (1:200), then incubated in a solution of peroxidase-labeled goat anti-rabbit Ab (1:1000). Bands were visualized by ECL. The arrows point to ß, 2 and 1 chains of Hp. This figure represents the results of three experiments.

Because Hp is stored in neutrophils within a cytoplasmic granular compartment (24) and the level of Hp is reduced in neutrophil extracts after stimulation of cells by TNF- as revealed by Western blot analysis, we attempted to evaluate by immunofluorescence the intracellular Hp content in unstimulated neutrophils or cells stimulated by TNF-. Hp was detected in the cytoplasm of >80% of purified neutrophils from cultures incubated with HBSS. Pretreatment of neutrophils with 13 ng/ml of TNF- for 1 h reduced the staining of the cells to a significant extent, leaving only about 10% of neutrophils stained with anti-Hp Ab (Table I). Fig. 4 shows the presence of Hp in intact neutrophils (green color) and its absence in TNF-stimulated cells.

View this table: [in this window] [in a new window]   Table I. Enumeration of cultured human polymorphonuclear neutrophils containing Hp by immunofluorescence before and after stimulation by TNF-1

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Immunocytochemical staining of Hp in control and TNF- stimulated human neutrophils. Human neutrophils (40 x 106/ml) were incubated either with HBSS (A) or with 1.3 ng/ml of TNF- (B) for 1 h at 37°C. The cells were then layered on glass slides and fixed in a solution of formaldehyde, methanol, and acetone (2:19:19 v/v) as described in Materials and Methods. After blocking of nonspecific binding with 1% BSA and 10% normal goat serum, the neutrophils were stained with rabbit anti-human Hp Ab (1:100), then treated with biotin-labeled goat anti-rabbit Ab (1:1000), followed by FITC-conjugated streptavidin. The green color (FITC) indicated the presence of Hp within the neutrophils. Propidium iodide solution was used for the nucleus coloration. The slides were viewed under a fluorescence microscope. One representative experiment of eight is shown.

Differential effect of TNFR-specific mutants on the release of Hp from neutrophils

As neutrophils are known to express both types of TNFR (28), p55 and p75, which are linked to different signaling pathways (18), TNF- receptor-specific mutants were used to investigate the role that the two receptor types play in the release of Hp from neutrophils.

Neutrophils were pretreated for 30 min with either wild-type TNF- or TNF-specific mutants at concentrations ranging from 0.13 ng/ml to 13 ng/ml. As a control, the cells were preincubated with HBSS. The results of these experiments are illustrated in Fig. 5, where it can be seen that TNF- and TNFR-p55-specific mutant stimulate the release of Hp from neutrophils in a concentration-dependent manner. It can be also seen that a similar concentration-dependent stimulation of the release of Hp was observed when neutrophils were pretreated with the TNFR-p55-specific mutant, which showed a significant response even at the lowest concentration used (0.13 ng/ml). Because the TNFR-p75-specific mutant has a 10-times lower affinity than the wild-type TNF- (20), we compared the intensity of the bands obtained with a given concentration of wild type to the intensity obtained with a 10- and 100-fold higher concentration of the TNFR-p75 mutant. In contrast to the wild-type TNF- and the TNFR-p55-specific mutant, we did not detect any significant release of Hp using the TNFR-p75-specific mutant, even at the highest concentration tested (13 ng/ml).

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Concentration-dependent release of Hp from neutrophils stimulated by TNFR-specific mutants. Neutrophils (40 x 106/ml) were incubated for 30 min at 37°C with different concentrations (0.13 ng/ml to 13 ng/ml) of wild-type TNF- or TNFR-specific mutants: TNFR-p55 and TNFR-p75. The buffer solution alone, HBSS, a TNF diluent, was added to the control cells. The Hp contents of the supernatants was tested by Western blot analysis as described in Materials and Methods. The arrows show the ß-, 2-, and 1-chains of Hp. One representative experiment of five is shown.

Involvement of tyrosine kinase in Hp release from neutrophils stimulated by TNF-

Protein phosphorylation is crucially involved in controlling various cell responses to external stimuli. Because TNF- induces tyrosine phosphorylation in suspended as well as adherent human neutrophils and because tyrosine kinase links the stimulation by TNF- to tyrosine phosphorylation and to different functions of neutrophils (21), we hypothesized that tyrosine kinase inhibitors might influence TNF--induced Hp release. To test this hypothesis, neutrophils were preincubated with erbstatin (Sigma) at 10 µg/ml (a concentration previously found to be optimal for the inhibition of TNF--induced tyrosine phosphorylation in human neutrophils (23)) for 30 and 60 min before incubation with TNF- for 30 min. Erbstatin was chosen because it was found to be the most potent inhibitor of several tyrosine kinase inhibitors with respect to its ability to inhibit TNF--induced tyrosine phosphorylation. As a control, DMSO, the diluent of erbstatin, was added to neutrophils. DMSO on its own had no effect on Hp release. After 30 min of preincubation with erbstatin, the release of Hp from TNF--stimulated neutrophils was dramatically decreased, and after 60 min it was completely inhibited (Fig. 6).

View larger version (56K): [in this window] [in a new window]   FIGURE 6. Effect of erbstatin on Hp release from neutrophils stimulated by TNF-. Neutrophils (5 x 106/ml) were pretreated with 10 µg/ml of erbstatin for 60 min at 37°C before incubation with 13 ng/ml TNF- for 1 h. The Hp content in the supernatants of stimulated neutrophils was assayed by Western blot analysis as described in Materials and Methods. This figure represents the results from four experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The interaction between Hp and lymphocytes on the one hand (31), and TNF- and neutrophils on the other hand (as described herein), could represent two tightly linked steps in the cascade of the acute phase response.

Hp binds to human peripheral blood neutrophils via a specific membrane receptor (14). Adding Hp to culture medium together with stimulatory agents such as fMLP, arachidonic acid, and opsonized zymosan, specifically inhibits the neutrophil respiratory burst activity (14). Recently, it was reported that Hp is present in considerable quantities within neutrophils and monocytes; exogenous Hp is taken up by these phagocytic cells from the medium and is not synthesized de novo by these cells (16). mAb against native Hp labels 90% of neutrophils and some monocytes in a granular staining pattern, but do not recognize lymphocytes (16), a finding we have confirmed using a polyclonal Ab against Hp. In our immunofluorescence study, >80% of the neutrophils were stained under control conditions. Some stored Hp may be released from neutrophils during phagocytosis (16). The results presented here document an alternative, cytokine-induced release of Hp. Stimulation by TNF- reduced the Hp-specific staining of cultured neutrophils to a significant extent, decreased the amount of cell-associated Hp, and enhanced the appearance of Hp in the cell supernatants, leading us to the conclude that TNF- induces the release of Hp from neutrophils. The existence of a small fraction of purified neutrophils (<20%) that did not stain with anti-Hp could be explained by the possible prior activation of those neutrophils in the bloodstream or during the experimental manipulations, but could also represent a distinct subpopulation. In addition to TNF-, fMLP, a classical neutrophil agonist, also induced Hp release. The effects of other neutrophil agonists remain to be examined.

TNF- has been implicated in mediating various pathological conditions. Serum levels of TNF- are increased during infections, injury, cachexia, or autoimmune diseases (33) and have been reported to be as high as 1 ng/ml in the serum of patients suffering from severe malaria (34). While the release of Hp from neutrophils was seen at TNF- concentrations of 0.13 ng/ml and higher in our in vitro conditions, comparison with in vivo conditions cannot be made solely on the basis of concentration of soluble TNF- in the serum, because many other factors may be involved. There is also a possibility of local accumulation of TNF-. The estimation of the level of TNF- in serum of patients is based on measuring soluble TNF-. In this respect, it is relevant to note that the membrane-bound form of TNF- is also bioactive and confers some typical TNF- responses (35) that may bias estimates of its potency. Furthermore, the detection of TNF- by ELISA using mAb depends on the recognition of epitopes, some of which may be masked by soluble TNFR.

TNF- is a cytokine with a wide range of biological activities that are mediated through two different receptors: p55 and p75 (18). The intracellular domains of the p55 and p75 TNFR differ, suggesting the usage of different signal transduction pathways (36). Indeed TNF-p55 activation induces a variety of proinflammatory responses (18, 37, 38) including cytotoxicity, while TNF-p75 elicits a limited number of cellular responses (39, 40). However, the two receptors may functionally be connected in that the p75 receptor facilitates the triggering of the p55 receptor at low TNF concentrations (37). To understand the relative role of the two types of receptors in TNF--induced release of Hp from neutrophils, we used TNF mutants that selectively bind to the different TNF- receptors. The results showed that the release of Hp induced by TNF- in neutrophils is predominantly mediated by the p55 receptor.

As we have shown in Results, the TNFR-p55 mutant was found to be about equipotent to the wild-type TNF- in its ability to stimulate the secretion of Hp. In contrast, little or no Hp release was observed in response to the TNFR-p75 mutant, even at concentrations up to 100-fold higher than those of the TNFR-p55 mutant. The lack of Hp release from neutrophils incubated with the TNFR-p75 mutant cannot be ascribed to a lack of binding because it exhibits only a 10-fold lower affinity toward p75 as compared with wild-type TNF- (20). While these data strongly support a primary role for p55 in the mediation of the secretion of Hp by human neutrophils induced by TNF-, an indirect contribution of p75 cannot be excluded because TNF- has been shown to induce the formation of heterocomplexes of the two receptor types on the surface of intact cells (41). Moreover, Richter et al. (42) have shown that TNF--induced superoxide production in adherent human neutrophils involves both TNFR-p55 and TNFR-p75. The authors suggested that TNFR-p75 concentrates TNF- at the cells surface and delivers it to TNFR-p55, which mediates intracellular signaling. TNFR-p75 could play a similar role in our present observations.

The data described above suggest that neutrophils could promptly increase the level of Hp at sites of infection or injury, preceding the augmentation of Hp synthesis by hepatocytes stimulated by inflammatory cytokines. In contrast, because Hp has been shown to be taken up by neutrophils from media containing Hp in vitro (Ref. 16 and our unpublished observation), it is possible that neutrophils also could very rapidly diminish excessive levels of Hp, faster than it would be transported to and catabolized by hepatocytes. Both of these reactive processes, namely uptake and release of Hp at the afflicted site, probably happen in the tissue. One can speculate that primary uptake, i.e., the loading of Hp to be transported to an afflicted site, is a different mechanism that takes place in the circulation. Therefore, it would be interesting to examine how different experimental approaches may modulate these processes. An additional factor that should be considered is the effect of neutrophil adherence to extracellular matrix components of various cell types because these interactions are known to exert a profound influence on the functional responsiveness of these cells to various agonists, including TNF- (43).

Therefore, prompt release of Hp may allow neutrophils to function, in addition to the phagocytic activity, by direct destruction of the infectious organisms. For example, by binding hemoglobin, Hp can deny bacteria access to a source of iron and in this way inhibit their replication (44). Alternatively, Hp-hemoglobin complexes bound within the phagolysosomes of phagocytic cells may generate reactive oxygen products that may potentiate the intraphagosomal destruction of infectious organisms, a mechanism proposed to explain of the trypanocidal activity of Hp-related protein (45).

Hp released by neutrophils may also act in an autocrine fashion. Binding sites for Hp on neutrophils have been defined (14, 16), and it was shown that Hp interacts with the ß₂ integrin CD11b (15). Furthermore, Hp has been shown to modulate the functional responsiveness of human neutrophils to various agonists such as fMLP, arachidonic acid, and opsonized zymosan, as shown by the inhibition of mobilization of calcium and of the stimulation of the metabolic burst (14). The potential effects of Hp on the responses of neutrophils to other agonists have not been determined as of yet. In view of the known interactions between CD11b and Fc receptors (46), it appears to be of particular importance to examine the effects of Hp on the phagocytic and phagocytosis-related functions of neutrophils.

Borregaard et al. have shown that human neutrophils contain albumin in an intracellular compartment (secretory vesicles). Albumin was released during activation of neutrophils by inflammatory mediators fMLP, platelet-activating factor, and leukotriene B₄ (47). Our data show that neutrophils at inflammatory sites also release the biologically active molecule, Hp. Furthermore, we have identified a substance, TNF-, that mediates this effect. Once released, Hp could significantly influence the behavior of the surrounding cells and of neutrophils themselves and thus regulate the development and outcome of inflammatory and immune reactions. The selective binding of Hp to the B cell-specific lectin CD22 (32), which appears to be involved in the regulation of Ag-specific B cell responses (48), gives evidence for such a regulatory mechanism. Our data contribute to the growing understanding of the involvement of neutrophils not only in nonspecific defense mechanisms, but also in the initiation and control of the afferent limb of the immune response.

In conclusion, in the present study a new pathway of cytokine-cell interactions is pointed out. The stimulation of Hp release from human neutrophils by TNF- is a hitherto undescribed property of this pleiotropic cytokine. Its biological significance may lie in a very fast adjustment of Hp levels in the process of the APR.

	Acknowledgments

 
We thank Dr. Hansruedi Loetscher (Hoffman-La Roche, Basel, Switzerland) for providing the TNF mutants, Dr. Niklaus Dürmüller and Dr. David Dresser for significant discussion and critical reading of the manuscript, and Guy Desharnais for his assistance in some experiments.

	Footnotes

 
¹ This work was supported in part by the research center of the Pavillon Saint-Françoise d’Assise, Centre Hospitalier Universitaire de Québec, and by grants from the Medical Research Council and the Arthritis Society of Canada.

² Address correspondence and reprint requests to Dr. Nadia Berkova at her current address: Centre National de la Recherche Scientifique UP41–Université de Rennes 1, Biologie et Génétique du Dévelopment, Equipe Dévelopment Précose, Faculté de Médecine, 2 avenue du Professeur Léon Bernard, CS 34 317, 35043 Rennes Cedex, France.

³ Abbreviations used in this paper: APR, acute phase response; Hp, haptoglobin; ECL, enhanced chemiluminescence.

Received for publication October 22, 1998. Accepted for publication February 23, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Baumann, H., J. Gauldie. 1994. The acute phase response. Immunol. Today 15:74.[Medline]
2. Vassalli, P.. 1992. The pathophysiology of tumor necrosis factors. Annu. Rev. Immunol. 10:411.[Medline]
3. Matsushima, K., J. J. Oppenheim.. 1989. Interleukin 8 and MCAF: novel inflammatory cytokines inducible by IL-1 and TNF. Cytokine 1:2.[Medline]
4. Cassatella, M. A.. 1991. The production of cytokines by polymorphonuclear neutrophils. Immunol. Today 16:21.
5. Gaudry, M., O. Brégerie, V. Andrieu, J. El Benna, M. A. Pocidalo, J. Hakim. 1997. Intracellular pool of vascular endothelial growth factor in human neutrophils. Blood 90:4153.[Abstract/Free Full Text]
6. Dinarello, C. A., J. G. Cannon, S. M. Wolff. 1988. New concepts on the pathogenesis of fever. Rev. Infect. Dis. 10:34.[Medline]
7. Neuhaus, O. W., A. Liu. 1964. Biochemical significance of serum glycoproteins. III. Hepatic production of 1 and 2 globulins responding to injury. Proc. Soc. Exp. Biol. 117:244.
8. Louagie, H., J. Delanghe, I. Desombere, M. De Buyzere, P. Hauser, G. Leroux-Rocls. 1993. Haptoglobin polymorphism and the immune response after hepatitis B vaccination. Vaccine 11:1188.[Medline]
9. Bowman, B. H.. 1993. Haptoglobin. B. H. Bowman, ed. Hepatic Plasma Proteins 149. Academic Press, San Diego.
10. Snellman, O., B. Sylven. 1967. Haptoglobin acting as a natural inhibitor of cathepsin B activity. Nature 216:1033.[Medline]
11. Cid, M. C., D. S. Grant, G. S. Hoffman, R. Auerbach, A. S. Fauci, H. K. Kleinman. 1993. Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J. Clin. Invest. 91:977.
12. Hoffman, L. H., V. P. Winfrey, G. L. Blaeuer, G. E. Olson. 1996. A haptoglobin-like glycoprotein is produced by implantation-stage rabbit endometrium. Biol. Reprod. 55:176.[Abstract]
13. Berkova, N., A. Lemay, P. De Grandpré, S. Goupil, R. Maheux. 1997. Immunoblot detection of decreased antibodies to haptoglobin-like protein in the serum of infertile patients with or without endometriosis. Biol. Reprod. 57:178.[Abstract]
14. Oh, S. K., N. Pavlotsky, A. I. Tauber. 1990. Specific binding of haptoglobin to human neutrophils and its functional consequences. J. Leukocyte Biol. 47:142.[Abstract]
15. El Ghmati, S., E. Van Hoeyveld, J. Van Strijp, J. Ceuppens, E. Stevens. 1996. Identification of haptoglobin as an alternative ligand for CD11b/CD18. J. Immunol. 156:2542.[Abstract]
16. Wagner, L., A. Gessl, S. Baumgartner Parzer, W. Base, W. Waldhausl, M. S. Pasternack. 1996. Haptoglobin phenotyping by newly developed monoclonal antibodies: demonstration of haptoglobin uptake into peripheral blood neutrophils and monocytes. J. Immunol. 156:1989.[Abstract]
17. Old, L. J.. 1990. Tumor necrosis factor. B. Bonavida, and G. Granger, eds. Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy 1. Karger, Basel, Switzerland.
18. Tartaglia, L. A., D. V. Goeddel. 1992. Two TNF receptors. Immunol. Today. 13:151.[Medline]
19. Dobrynin, V., N. Berkova, E. Boldyreva, N. Bystrov, V. Kravchenko, S. Filippov, S. Chuvpilo, O. Shamborant, and V. Korobko. 1988. Expression in E. coli of artificial DNA coding for human tumor necrosis factor. Soviet J. Bioorg. Chem. 14:1530 (translated from Bioorg. Khim. (USSR) 14:1530).
20. Loetscher, H., D. Stueber, D. Banner, F. Mackay, W. Lesslauer. 1993. Human tumor necrosis factor- (TNF-) mutants with exclusive specificity for the 55-kDa or 75-kDa TNF receptors. J. Biol. Chem. 268:26350.[Abstract/Free Full Text]
21. Al-Shami, A., C. Gilbert, F. Barabé, M. Gaudry, P. Naccache. 1997. Preservation of the pattern of tyrosine phosphorylation in human neutrophils lysates. J. Immunol. Methods 202:183.[Medline]
22. Gomperts, B. D.. 1983. Involvement of guanine nucleotide-binding protein in the gating of Ca²⁺ by receptors. Nature 306:64.[Medline]
23. Naccache, P. H., C. Gilbert, A. Caon, M. Gaudry, C.-K. Huang, V. Bonak, K. Umezawa, S. McColl. 1990. Selective inhibition of human neutrophil functional responsiveness by erbstatin, an inhibitor of tyrosine protein kinase. Blood 76:2048.
24. Laemmli, U. K.. 1970. Cleavage of structural proteins of the head of the bacteriophage T4. Nature 277:680.
25. Manes, M., G. Bonfant, P. Belfani, A. M. Gaiter, S. Alloatti. 1997. Diagnostic aspects in the determination of antineutrophil cytoplasmic antibodies. Recent Prog. Med. 88:17.
26. Hagen, E. C., B. E. Ballieux, L. A. van Es, M. R. Daha, F. J. van der Woude. 1993. Antineutrophil cytoplasmic autoantibodies: a review of the antigens involved, the assays, and the clinical and possible pathogenetic consequences. Blood 81:1996.[Free Full Text]
27. Sternberger, L. A.. 1979. Immunofluorescence. L. A. Sternberger, ed. Immunocytochemistry Second Edition.32. John Wiley & Sons, New York.
28. Gon, S., T. Gatanaga, F. Sendo. 1996. Involvement of two types of TNF receptor in TNF- induced neutrophil apoptosis. Microbiol. Immunol. 40:463.[Medline]
29. Barclay, R.. 1985. The role of iron in infection. Med. Lab. Sci. 42:166.[Medline]
30. Kohler, W., O. Prokop. 1978. Relationship between haptoglobin and Streptococcus pyogenes T4 antigens. Nature 271:373.[Medline]
31. Langlois, M. R, J. R. Delanghe. 1996. Biological and clinical significance of haptoglobin polymorphism in humans. Clin. Chem. 42:1589.[Abstract/Free Full Text]
32. Hanasaki, K., J. D. Powell, A. Varki. 1995. Binding of human plasma sialoglycoproteins by the B cell-specific lectin CD22. J. Biol. Chem. 270:7543.[Abstract/Free Full Text]
33. Tracey, K. J., A. Cerami. 1991. Pleiotropic effects of TNF in infection and neoplasia: beneficial, inflammatory, catabolic, or injurious. B. Aggarwal, and J. Vilcek, eds. Tumor Necrosis Factors: Structure, Functions, and Mechanism of Action 431. Marcel Dekker, New York.
34. Kern, P., C. Hemmer, J. Van Damme, H. Gruss, M. Dietrich. 1989. Elevated tumor necrosis factor and interleukin-6 serum levels as markers for complicated Plasmodium falciparum malaria. Am. J. Med. 87:139.[Medline]
35. Grell, M., E. Douni, H. Wajant, M. Lohden, M. Clauss, B. Maxeiner, S. Georgopoulos, W. Lesslauer, G. Kollias, K. Pfizenmaier, P. Scheurich. 1995. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell 83:793.[Medline]
36. Loetscher, H., E. J. Schlaeger, H. W. Lahm, Y. C. E. Pan, W. Lesslauer, M. Brockhaus. 1990. Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells. J. Biol. Chem. 265:20131.[Abstract/Free Full Text]
37. Tartaglia, L. A., T. M. Ayres, G. H. W. Wong, D. V. Goeddel. 1993. A novel domain within the 55 kd TNF receptor signals cell death. Cell 74:845.[Medline]
38. Barbara, J. A. J., W. B. Smith, J. R. Gamble, X. van Ostade, P. J. Tavernier, W. Fiers, M. A. Vadas, A. F. Lopez. 1994. Dissociation of TNF- cytotoxic and proinflammatory activities by p55 receptor and p75 receptor-selective TNF- mutants. EMBO J. 13:843.[Medline]
39. Tartaglia, L. A., D. V. Goeddel, C. Reynolds, I. S. Figari, R. F. Weber, B. M. Fendly, M. A. Palladino. 1993. Stimulation of human T-cell proliferation by specific activation of the 75 kDa tumor necrosis factor receptor. J. Immunol. 151:4637.[Abstract]
40. Zheng, L., G. Fisher, R. E. Miller, J. Peschon, D. H. Lynch, M. J. Lenardo. 1995. Induction of apoptosis in mature T cells by tumour necrosis factor. Nature 377:348.[Medline]
41. Pinckard, J. K., K. C. F. Sheehan, R. D. Schreiber. 1997. Ligand-induced formation of p55 and p75 tumor necrosis factor receptor heterocomplexes on intact cells. J. Biol. Chem. 272:10784.[Abstract/Free Full Text]
42. Richter, J., U. Gullberg, M. Lantz. 1995. TNF-induced superoxide anion production in adherent human neutrophils involves both the p55 and p75 TNF receptor. J. Immunol. 154:4142.[Abstract]
43. Nathan, C. F.. 1987. Neutrophil activation of biological surfaces: massive secretion of hydrogen peroxidase in response to products of macrophages and lymphocytes. J. Clin. Invest. 80:1550.
44. Eaton, J. W., P. Brandt, J. R. Mahoney, J. T. Lee. 1982. Haptoglobin: a natural bacteriostat. Science 215:284.[Free Full Text]
45. Smith, A. B., J. D. Esko, S. L. Hajduk. 1996. Killing of trypanosomes by the human haptoglobin-related protein. Science 268:284.
46. Galon, J., J. F. Gauchat, N. Mazières, R. Spagnoli, W. Storkus, M. Lötze, J. Y. Bonnefoy, W. H. Fridman. 1996. Soluble Fc receptor type III (FcRIII, Cd 16) triggers cell activation through interaction with complement receptors. J. Immunol. 157:1184.[Abstract]
47. Borregaard, N., L. Kjeldsen, K. Rygaard, L. Bastholm, M. Nielsen, H. Sengelov, O. W. Bjerrum, A. Johnsen. 1992. Stimulus-dependent secretion of plasma proteins from human neutrophils. J. Clin. Invest. 90:86.
48. Clark, E. A.. 1993. CD22, a B cell-specific receptors, mediates adhesion and signal transduction. J. Immunol. 150:4715.[Medline]

This article has been cited by other articles:

				J. N. Fain, S. W. Bahouth, and A. K. Madan Haptoglobin release by human adipose tissue in primary culture J. Lipid Res., March 1, 2004; 45(3): 536 - 542. [Abstract] [Full Text] [PDF]

				N. Berkova, A. Lemay, D. W. Dresser, J.-Y. Fontaine, J. Kerizit, and S. Goupil Haptoglobin is present in human endometrium and shows elevated levels in the decidua during pregnancy Mol. Hum. Reprod., August 1, 2001; 7(8): 747 - 754. [Abstract] [Full Text] [PDF]

				M. Piva, G. M. Horowitz, and K. L. Sharpe-Timms Interleukin-6 Differentially Stimulates Haptoglobin Production by Peritoneal and Endometriotic Cells in Vitro: A Model for Endometrial-Peritoneal Interaction in Endometriosis J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2553 - 2561. [Abstract] [Full Text] [PDF]

				K.L. Sharpe-Timms, E.A. Ricke, M. Piva, and G.M. Horowitz Differential expression and localization of de-novo synthesized endometriotic haptoglobin in endometrium and endometriotic lesions Hum. Reprod., October 1, 2000; 15(10): 2180 - 2185. [Abstract] [Full Text] [PDF]

				M. R. Langlois, M.-E. Martin, J. R. Boelaert, C. Beaumont, Y. E. Taes, M. L. De Buyzere, D. R. Bernard, H. M. Neels, and J. R. Delanghe The Haptoglobin 2-2 Phenotype Affects Serum Markers of Iron Status in Healthy Males Clin. Chem., October 1, 2000; 46(10): 1619 - 1625. [Abstract] [Full Text] [PDF]

				F. Yang, A. J. Ghio, D. C. Herbert, F. J. Weaker, C. A. Walter, and J. J. Coalson Pulmonary Expression of the Human Haptoglobin Gene Am. J. Respir. Cell Mol. Biol., September 1, 2000; 23(3): 277 - 282. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Berkova, N. Articles by Naccache, P. H. Search for Related Content PubMed PubMed Citation Articles by Berkova, N. Articles by Naccache, P. H.