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(Journal of Leukocyte Biology. 2001;70:59-64.)
© 2001 by Society for Leukocyte Biology

Dimeric S100A8 in human neutrophils is diminished after phagocytosis

Rakesh K. Kumar*, Zheng Yang*, Susan Bilson*, Soula Thliveris*, Bridget E. Cooke{dagger} and Carolyn L. Geczy*

* School of Pathology, University of New South Wales, Sydney, Australia; and
{dagger} Department of Anatomical Pathology, Prince of Wales Hospital, Randwick, Australia

Correspondence: R. K. Kumar, School of Pathology, University of New South Wales, Sydney, Australia 2052. E-mail: R.Kumar{at}unsw.edu.au


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ABSTRACT
 
S100A8 is a major cytoplasmic protein of neutrophils and monocytes/macrophages and has been associated with myeloid cell differentiation and activation. Little is known about its functions or mechanisms of release from neutrophils. We have developed a monoclonal antibody to murine S100A8, which cross-reacts with human S100A8. This antibody, which recognizes the homodimeric form of the protein, detects its expression specifically in human neutrophils and is reactive in formalin-fixed, paraffin-embedded tissues. Using this antibody as well as a commercially available antibody to human S100A8, we show that phagocytic activation of neutrophils, in vivo in acute appendicitis and in vitro following phagocytosis of opsonized zymosan, is characterized by loss of cytoplasmic immunoreactivity for S100A8. In vitro, phagocytosis is associated with rapid diminution of immunostaining without loss of viability. Loss of immunoreactivity for S100A8 may serve as a marker of localized neutrophil activation in tissues.

Key Words: acute inflammation • neutrophil activation • immuno-histochemistry • S100 proteins


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INTRODUCTION
 
The S100 proteins designated S100A8 and S100A9 have been detected in the cytoplasm of neutrophils, monocytes, and tissue macrophages at sites of inflammation. These proteins are believed to be present as noncovalent heterodimers, which constitute approximately 40% of the cytosolic protein of neutrophils [1 ]. Intracellular functions are still incompletely understood [2 ] but include a role in regulation of neutrophil activation involving Ca2+-dependent signaling [3 ] and involvement in transporting unsaturated fatty acids [4 ]. Extracellular S100A9 has anti-microbial activity, possibly by virtue of its ability to chelate zinc [5 ]. A9 may also modulate the affinity of integrins expressed on neutrophils [6 ], and A8 inhibits this activity [6 ]. S100A9 inhibits the oxidative burst of bacillus Calmette-Guerin (BCG)-activated macrophages and suppresses inflammatory pain [7 , 8 ].

High levels of extracellular A8/A9 complex are suggested markers of disease activity in inflammatory disorders such as arthritis and ulcerative colitis [9 , 10 ]. Whether the complex is released by dying cells [11 ] or its presence is a result of active secretion remains unclear. The proteins lack signal sequences associated with classical secretory pathways, although a tubulin-dependent mechanism of secretion by monocytes is suggested [12 ]. There is no information available about mechanisms of secretion of A8 and/or A9 by neutrophils.

Our laboratory has a long-standing interest in a murine S100 protein, originally designated CP-10 [13 ], the nearest human counterpart to which is S100A8 [14 ]. Unlike human S100A8, from which it exhibits substantial sequence divergence, CP-10 is a potent chemoattractant for phagocytic cells [15 ]. However, like the human protein, it is expressed in high amounts by neutrophils and by activated macrophages [16 , 17 ] so that it has come to be known as murine S100A8 (mA8) [18 ]. Previously, we demonstrated that in murine experimental-acute alveolitis induced by bleomycin, large quantities of this protein are released into the extracellular fluid in a biologically active, monomeric form, apparently by recruited neutrophils [19 ]. In the present study, we have used antibodies to human and murine S100A8 to examine the expression of the A8 protein by human neutrophils, in vivo and in vitro. We show for the first time that homodimeric S100A8 is present in the cytoplasm of human neutrophils and that following phagocytosis, neutrophils exhibit rapid loss of cytoplasmic immunoreactivity for homodimeric S100A8, which may serve as a marker of localized neutrophil activation in tissues.


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MATERIALS AND METHODS
 
Preparation of a rat monoclonal antibody (mAb) to mA8
Sprague-Dawley rats, aged 3–4 weeks (Animal Resources Centre, Perth, Western Australia), were immunized on days 0, 9, and 14 with 7–10 µg bacterial fusion protein of mA8 with glutathione S-transferase (GST) bound to glutathione-agarose beads. Popliteal lymph-node cells from immunized animals were harvested, mixed with myeloma cells (P3-X63-Ag8.653) at a ratio of 2.5:1 [20 ], and fused in 50% polyethylene glycol (Merck 4000, Merck, Rahway, NJ). Cells were resuspended in a mixture of RPMI 1640 medium, 20% fetal calf serum, 40% rat thymocyte-conditioned medium, and 1% hypoxanthine-aminopterin-thymidine, then dispensed into 96-well plates, and pre-seeded with macrophage feeder cells. Hybridoma supernatants were screened 12–21 days post-fusion with an indirect enzyme immunoassay using plates coated with 500 ng/well of CP-10/GST fusion protein or GST alone. Of the 131 hybridomas tested, 13 positive clones were detected, which were unreactive to GST, and of these, five were re-cloned twice after selecting for immunoglobulin G (IgG)-positive clones. The mAb designated CP10.1 was selected for further use, based on its high immunoreactivity with native and recombinant mA8 as well as with relevant target cells. Because the hybridoma could not be grown as an ascites in mice or rats, cells were maintained in appropriately supplemented HY medium (Sigma Chemical Co., St. Louis, MO).

Immunoblotting
Recombinant S100A8, S100A9, or S100A12 (2 µg) purified from C4 reverse-phase HPLC [21 ] was lyophilized, resuspended in 50 mM Tris-HCl, pH 7.5, and oxidized in 2 mM copper sulfate at room temperature for 30 min. The formation of homo- or heterodimers was confirmed by polyacrylamide gel electrophoresis (PAGE) and silver staining. Samples were mixed with an equal volume of 2x loading buffer, heated at 100°C for 3 min, and analyzed by 10% tricine sodium dodecyl sulfate (SDS)-PAGE [22 ]. Gels run in parallel were silver stained or transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Antigenic reactivity of the S100 protein monomers and dimers to the rat mAb CP10.1 or the 85-C2 mouse mAb (BMA Biomedicals AG, Augst, Switzerland) was detected using horseradish peroxidase (HRP)-conjugated, goat anti-mouse or anti-rat IgG (Bio-Rad, Hercules, CA), respectively, and visualized by chemiluminescence [enhanced chemiluminescence (ECL) system, Amersham Australia, Sydney, Australia].

Immunostaining
Immunostaining was performed using a capillary-action staining system as described previously [19 ]. Cells expressing cytoplasmic immunoreactivity for S100 A8 were identified using CP10.1 or 85-C2. For sections of archival, formalin-fixed blocks of tissue from tuberculous lymph nodes and acute appendicitis, antigen retrieval was performed by boiling in 0.01 M citrate buffer, pH 6.0, for 10 min. Sections were washed with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 and blocked with 50% normal rabbit serum for 10 min at 37°C. They were then incubated for 30 min at 37°C with an appropriate concentration of the primary antibody. Sections were washed four times with PBS-Triton after this and each subsequent incubation. Bound antibody was detected by sequential, 30-min incubations with biotinylated rabbit anti-rat or anti-mouse IgG (Dako, Sydney, Australia), followed by a complex of streptavidin (Boehringer Mannheim, Sydney, Australia) and biotinylated HRP in PBS-1% bovine serum albumin (BSA). Diaminobenzidine was used as the substrate, and sections were counterstained lightly with hematoxylin. For cytospin preparations, a similar procedure was used, but antigen retrieval was not required, and nuclei were counterstained with methyl green.

Enzyme immunoassay
Wells of a microtitre plate (Nunc, Roskilde, Denmark) were coated with 500 ng recombinant human or murine S100A8 (prepared according the protocols described in ref. [16 ] and comprising a mixture of monomeric and dimeric forms) in carbonate-bicarbonate buffer, pH 9.6, for 2 h at 37°C, washed four times with Tris-buffered saline (TBS; pH 7.4) containing 0.01% Tween-20, blocked with TBS-Tween containing 0.1% ovalbumin for 60 min at 37°C, and washed again. To compare the binding of CP10.1 with the two forms of S100A8, serial dilutions of mAb supernatant were added to the wells in triplicate and incubated overnight at 4°C. To assess competitive inhibition, a 1:25 dilution of CP10.1 antibody was added to each well of a plate coated with human S100A8 in the presence of 0.05–0.5 µg/ml recombinant murine or human S100A8. The wells were then washed four times, and bound antibody was detected by incubation for 60 min at room temperature with biotinylated rabbit, anti-rat IgG, followed by additional washes and incubation with streptavidin-HRP complex (Amersham Australia). Enzyme activity was revealed by incubation with ABTS substrate (Kirkegaard & Perry, Gaithersburg, MD), and the reaction product was read using a Titertek Multiskan photometer with a 405 nm filter.

Stimulation of neutrophils in vitro
Freshly collected blood samples were layered onto Mono-Poly Resolving medium (Ficoll-Hypaque, ICN Biomedicals, Aurora, OH), according to the manufacturer’s instructions and centrifuged at 600 g for 1 h. Neutrophils removed from the interface were washed twice and resuspended in RPMI 1640 at 5 x 106 cells/ml.

Neutrophils were exposed to N-formyl-Met-Leu-Phe (fMLP; 10-7 M; Sigma) or to opsonized zymosan for 15 min or 2 h. Opsonized zymosan was prepared according to the method of Markert et al. [23 ] and used at dilutions ranging from 1:1–1:20. After incubation, aliquots of the neutrophil suspension were removed for assessment of viability using trypan blue exclusion and for preparation of cytospin slides. The latter were air-dried, fixed in 2% paraformaldehyde for 30 min, rinsed in PBS for at least 5 min, and stored at 4°C.


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RESULTS
 
Specificity of CP10.1 antibody
Immunblotting demonstrated that mAb CP10.1 reacted with human S100A8 but not with S100A9 or with S100A12 (Fig. 1 ). Of interest was the observation that whereas the mAb 85-C2 detected monomeric and dimeric forms of S100A8, CP10.1 only reacted with the homodimer.



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  		Figure 1. Immunoblotting of monomeric and dimeric forms of S100A8, S100A9, and S100A12 using CP10.1 and 85-C2 mAbs reacted against monomeric S100A8 (A8m), homodimeric S100A8 (A8d), monomeric S100A9 (A9m), homodimeric S100A9 (A9d), a 1:1 mixture of oxidized S100A8 and S100A9 (A8/9), or S100A12 (A12). (A) 85-C2 reacts with monomeric and dimeric forms of S100A8 but not with S100A9 or S100A12. (B) CP10.1 reacts only with homodimeric S100A8 and exhibits no cross-reactivity with S100A9 or S100A12. (C) The corresponding silver-stained gel shows the migration patterns of the S100 proteins. Note that A8 + 9 contains homodimeric A8 (20 kDa), A8/A9 heterodimeric complex (24 kDa), and homodimeric A9 (28 kDa).

CP10.1 reactivity with human S100A8 was confirmed by titration and cross-absorption experiments. Using an enzyme immunoassay in which human or murine S100A8 was immobilized onto a plastic surface, comparable binding of CP10.1 to both proteins was demonstrated (Fig. 2 ). Furthermore, binding to human S100A8 was inhibited specifically by increasing concentrations of human or murine S100A8 (with >90% inhibition of binding of a 1:25 dilution by 100 ng/ml of either protein).



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  		Figure 2. Binding serial dilutions of CP10.1 antibody to wells coated with 500 ng recombinant human or murine S100A8 or to control wells. Values are optical density at 405 nm (mean±SE of triplicate samples).

Cellular immunolabeling with CP10.1 antibody
The 85-C2 mAb to human S100A8 strongly labeled neutrophils and macrophages in tissue sections of human lymph node (Fig. 3A ). In contrast, CP10.1 antibody generated against murine S100A8 appeared to label only neutrophils (Fig. 3B) . This difference in specificity was obvious, particularly when immunostaining sections of granulomas in tuberculous lymph nodes in which modified macrophages, including morphologically distinctive epithelioid and giant cells, were labeled by 85-C2, whereas only neutrophils in the caseous necrotic center were recognized by CP10.1 (Fig. 3C and 3D) .



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  		Figure 3. Immunolabeling leukocytes by antibodies to S100A8 in sections of human lymph node involved by tuberculous lymphadenitis. (A) Using 85-C2 antibody, reactivity of intravascular neutrophils and tissue macrophages in subcapsular tissue is apparent, whereas in B, only neutrophils are labeled by CP10.1 antibody. (C) 85-C2 antibody labels epitheloid cells in a tuberculous granuloma, as well as neutrophils in the caseous necrotic center, and in D, only neutrophils exhibit immunoreactivity with CP10.1 antibody.

Immunostaining neutrophils in tissues by CP10.1 was blocked by excess human or murine S100A8 (unpublished results). It is interesting that immunostaining by 85-C2 was blocked by human but not by murine S100A8.

Immunolabeling neutrophils in acute appendicitis by CP10.1
Using a polyclonal antibody to S100A8, we showed that in an experimental inflammatory lesion in mice, neutrophils were apparently activated to release S100A8, with loss of immunoreactivity in tissue sections [19 ]. To investigate whether a similar process occurred in human inflammatory lesions, sections of acute appendicitis were immunostained for S100A8 using CP10.1 antibody. An obvious gradation of intensity of immunostaining of neutrophils was observed from the serosa to the lumen. Intense staining of cells was noted in the serosa, where the neutrophils are recruited as part of an exudative inflammatory response (Fig. 4A ). Staining intensity diminished through the muscularis and the mucosa, which are sites of bacterial invasion in established appendicitis (Fig. 4B and 4C) . In the lumen, which contained large numbers of neutrophils participating in the suppurative inflammatory response, virtually none of the cells exhibited positive staining (Fig. 4D) . Identical results with respect to immunostaining of neutrophils were obtained with the 85-C2 antibody (unpublished results).



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  		Figure 4. Immunolabeling neutrophils for S100A8 in various layers of the appendix in acute appendicitis, using CP10.1 antibody. (A) Intense reactivity of neutrophils in the serosa is obvious, whereas in B, the staining of neutrophils in between the smooth-muscle fibersis less marked. Neutrophils infiltrating the mucosal layer in C are still less strongly reactive, and the cells in the (D) lumen exhibit no immunostaining for S100A8.

Immunolabeling neutrophils by CP10.1 after stimulation in vitro
Next, we investigated whether the loss of immunoreactivity of neutrophils for S100A8 at an inflammatory site in vivo could be triggered by exposure to bacterial products or required activation by phagocytosis. Following activation with fMLP, isolated neutrophils remained strongly immunoreactive for S100A8 with CP10.1 and 85-C2 mAbs and were indistinguishable from the unstimulated, control cells (Fig. 5A ). In contrast, neutrophils that had phagocytozed opsonized zymosan demonstrated markedly diminished immunoreactivity after 15 min of incubation, with a proportion exhibiting no staining at all (Fig. 5B) . This loss of immunostaining was observed using both mAbs. It is important that cells that had reduced immunoreactivity exhibited no evidence of selective loss of viability as estimated by trypan blue exclusion (Table 1 ). Indeed, the viability of all three preparations was virtually identical after 15 min, by which time loss of immunoreactivity was apparent already. After 2 h, only the cells exposed to fMLP showed any noticeable decrease in viability relative to controls, yet these cells were still strongly immunoreactive for S100A8.



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  		Figure 5. Isolated human neutrophils immunolabeled for S100A8 using CP10.1 antibody. (A) Neutrophils exposed to 10-7 M fMLP remained strongly immunoreactive. (B) Many of the neutrophils that had phagocytosed opsonized zymosan exhibited markedly diminished reactivity (e.g., vertical arrow) with some cells completely failing to stain for S100A8 (horizontal arrow).


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  		Table 1. Viability of Isolated Human Neutrophils after Stimulation In vitro


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DISCUSSION
 
In this study, we show that homodimeric S100A8 is present in the cytoplasm of human neutrophils. Activated neutrophils lose immunoreactivity for S100A8, in vitro and in vivo, and apparently, this is related to phagocytosis. In vitro, loss of cytoplasmic immunostaining is not associated with any reduction in viability.

Demonstration of changes in cytoplasmic immunoreactivity of neutrophils was facilitated by a novel mAb, which we designated CP10.1, raised against murine S100A8 and cross-reactive with human S100A8. This exhibited specific staining of the protein in human neutrophils. In earlier studies, there has been conflicting information about patterns of immunostaining for S100A8 in different inflammatory lesions (e.g., refs. [24 , 25 ]). However, most such antibodies were poorly characterized in terms of epitopes recognized. Variability in staining patterns may be a consequence of cross-reactivity with other S100 proteins, which is likely in view of their high degree of sequence homology, particularly in the calcium-binding and N-terminal domains [26 ]. At least some mAbs to S100A8 cross-react with S100A9 [27 ], and others recognize the A8/A9 complex preferentially [28 ]. Among antibodies specific for S100A8, different commercially available antibodies apparently recognize different epitopes on S100A8, because some permit demonstration of immunoreactivity in formalin-fixed, paraffin-embedded tissue (with or without antigen retrieval), whereas others do not.

However, differential staining of neutrophils and monocytes by an antibody to S100A8 has not been shown previously. The CP10.1 antibody specifically recognizes an epitope on homodimeric S100A8, which is evidently present on human and murine S100A8, is distinct from the epitopes recognized by other antibodies, and is expressed only by the protein in neutrophils. Of particular value for immunocytochemistry is the preservation of the neutrophil-specific epitope in paraffin-embedded tissues. Thus, the antibody to murine S100A8 provides a unique reagent with which to investigate the expression of this protein by human neutrophils.

In acute appendicitis, the inflammatory response is triggered by bacterial proliferation within the lumen, with subsequent spread of organisms through the wall of the appendix leading to transmural inflammation. Therefore, neutrophils in the lumen are virtually certain to have encountered and phagocytosed bacteria, whereas those in the serosal layers are less likely to have participated in a phagocytic response. In this context, the differential intensity of staining for S100A8 of neutrophils in the various layers of the appendicular wall could be explained in terms of phagocytic activation following interaction with the bacteria. Whether this represents extracellular release of the protein or alteration of the intracellular protein leading to loss of immunoreactivity is unclear. Release of neutrophil S100A8 in response to bacterial infection has not been demonstrated in human disease, although previously, we have shown high levels of S100A8 in murine abscess fluid [29 ].

To ascertain whether the loss of immunoreactivity was more likely to have been elicited by exposure to bacterial products or by the phagocytic response, we examined neutrophils activated by the potent bacterial chemoattractant fMLP, compared with cells that had phagocytosed particles of opsonized zymosan. There was no alteration in immunostaining following exposure to an optimal chemoattractant concentration of fMLP, whereas reactivity was reduced rapidly and strikingly after phagocytosis. This suggests that phagocytosis is an important trigger for the diminution of cytoplasmic immunoreactivity for S100A8, although it is unlikely to be the only one, because previously, we have demonstrated loss of neutrophil immunoreactivity for S100A8 in a model of alveolitis in which no particulate irritant was present [19 ]. The reduction in immunostaining was not merely a consequence of leakage of the protein from dead or dying cells, because there was no evidence of any selective decrease in neutrophil viability after engulfment of particles. Thus, it is possible that the loss of immunoreactivity might be a consequence of release of S100A8 by active secretion. However, we were unable to demonstrate convincingly increased levels of S100A8 in cellular supernatants commensurate with the loss of immunoreactivity by immunoblotting or enzyme immunoassay, although clearly, A8 and A9 were present in the supernatants (unpublished results).

Earlier studies demonstrate Ca2+-dependent translocation of the S100A8/A9 complex to the membrane of neutrophils activated with fMLP or activated zymosan [3 , 30 ], and these proteins are postulated to regulate Ca2+-dependent processes important in neutrophil migration, phagocytosis, and activation. Guignard et al. [31 ] show that maximal A8/A9 translocation was provoked by zymosan, whereas that by fMLP was relatively weak. The mechanism by which cytoplasmic immunoreactivity for A8 is lost is unclear. One possibility is that it might be related to alteration of protein conformation or redox state, e.g., by hypochlorite generated by activated neutrophils, so that the epitope recognized by the CP10.1 antibody on homodimeric S100A8 is no longer available. However, immunostaining using the 85-C2 antibody was diminished in parallel, in vitro and in vivo, suggestive of extracellular release of the protein. In any case, loss of immunoreactivity for S100A8 appears to be a marker of phagocytically activated neutrophils.

If neutrophil S100A8 is released, it would be of interest to establish whether this occurs as the free protein (monomeric or oligomeric) or in association with S100A9. In parallel experiments using a polyclonal antibody to human S100A9, we noted comparable loss of immunostaining for A9 (unpublished results), consistent with release of the complex. An important and as yet unresolved issue is the function of S100A8 following release. In the light of studies of anti-microbial activity of the A8/A9 complex and of zinc sequestration by A9 in the complex [5 ], release in response to phagocytosis of bacteria could be important in microbial defense. Of particular interest, however, is that S100A8 can be oxidized rapidly by hypochlorite to yield novel oxidation products ([32 ] and unpublished results), and an intriguing possibility is that it may minimize nonspecific tissue damage by neutrophil-derived, reactive oxygen species. Clearly, further investigation of these issues is needed.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Health and Medical Research Council of Australia and by the Community Health and Anti-Tuberculosis Association.

Received June 19, 2000; revised February 21, 2001; accepted February 22, 2001.


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