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Proteases produced by activated neutrophils are able to release soluble CD23 fragments endowed with proinflammatory effects ¹

INSERM U343, Hôpital de L’Archet, F-06202 Nice cedex 3, France

³Correspondence: INSERM U343, Hôpital de l’Archet, B.P. 79, F-06202 Nice cedex 3, France. E-mail: cousin{at}unice.fr

SPECIFIC AIMS

We investigated the capacity of polymorphonuclear neutrophil (PMN) -derived proteases to cleave CD23 (FcRII) from cell surface and addressed the effects of the released fragments on inflammatory process.

PRINCIPAL FINDINGS

1. Cleavage of membrane CD23 by serine proteases expressed on activated neutrophils
B cells expressing CD23 (the low-affinity receptor for immunoglobulin E, FcRII) were cocultured with varying numbers of f-MLP- or PDBu-activated blood PMN. A diminution of membrane CD23 (mCD23) expression was correlated with an increasing number of activated PMN (Fig. 1A ). The decrease in mCD23 expression was completely reversed by serum containing several protease inhibitors, synthetic, or physiological serine protease inhibitors (PMSF or 1-antitrypsin) (Fig. 1B ). When the coculture was performed in the presence of an HLE-specific inhibitor (methoxysuccinyl-AAPA chloromethylketone), a significant but partial inhibition was obtained. On the contrary, the metalloprotease inhibitor BB94, which has been described to inhibit the spontaneous shedding of CD23, had no effect on the diminution of CD23 expression induced by PMN (Fig. 1B ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Cocultures of PMN and B cells. A) Increasing numbers of PMN (0, 0.1, 0.25, 0.5x106/sample) activated by f-MLP (gray bars) or PDBu (hatched bars) were added to the SKW6.4 B cell line (0.5x106/sample). After 90 min, mCD23 expression on B cells was analyzed by flow cytometry using a CD23 mAb (FITC-mAb25). The results are expressed as percentages relative to MFI of SKW6.4 cells cultured without PMN and are the mean ± SD of three independent experiments. B) Stimulated PMN were preincubated alone or with autologous serum (10%), 1-AT (1, 50 µg/ml) or PMSF (1 mM), elastase inhibitor II (EI, 50 µg/ml), or BB94 (10 µM), then added to SKW6.4 cells at a cellular ratio of 0.5 (0.25x106 PMN:0.5x106 B cells). Results are expressed as in panel A.

2. Cleavage of membrane CD23 by purified human leukocyte elastase or cathepsin G
Since human leukocyte elastase (HLE) and cathepsin G (cG) remain bound to the surface of activated PMN, we used purified enzymes to further characterize the activities responsible for the decrease in mCD23 expression. When B cells were incubated with HLE for 2–1/2 h, diminution of mCD23 was obtained in a dose-dependent manner with an half-maximal effect of 0.32 ± 0.09 µg/ml (mean±SD of four independent experiments). The ability of HLE to cleave recombinant soluble CD23 demonstrated a direct proteolytic effect of HLE on CD23. To assess the specificity of CD23 cleavage by HLE, we investigated the sensitivity to HLE of other membrane proteins expressed on B cells such as CD19, CD20, surface Ig, HLA class I, and HLA-DR. Notably, these proteins were not affected, even at a concentration much higher than that sufficient to totally cleave CD23. The cG was also able to cleave CD23 from B cells surface in a dose-dependent manner. Moreover, CD23 receptor was shed by these two serine proteases from the surface of a monocytic cell line as well as B cells.

3. Fragments released by HLE
By immunoblotting and ELISA analysis, we show that soluble CD23 (sCD23) fragments released by HLE or activated PMN were mainly short fragments of 25 kDa or smaller.

4. Activation of monocytes by sCD23 fragments released by HLE
The 25 kDa recombinant sCD23 has been shown to activate human monocytes by interacting with CD11b,c/CD18 or the complex vitronectin receptor/CD47. We investigated a possible effect of sCD23 fragments released by HLE or cG on these cells. Supernatants from protease-treated CD23⁺ or CD23^- Daudi cells were added to freshly isolated monocytes without any costimulatory signal. We evaluated membrane CD14 expression (mCD14), oxidative burst, and secretion of tumor necrosis factor (TNF-). Stimulation of monocytes with HLE-released sCD23 up-regulated mCD14 by 55 ± 8% and ROS (radical oxygen species) production by 30 ± 6% (mean of percentages of increase ± SD obtained with CD23⁺ cell supernatant vs. CD23^- cell supernatant, n=6). Similar results were obtained when sCD23 from cG-treated CD23⁺ Daudi cells were used to stimulate monocytes. In addition, andas illustrated in Fig. 2 , sCD23 released by HLE or cG induced the secretion of TNF- (Fig. 2) . As expected, sCD23-mediated mCD14 up-regulation, ROS production and TNF- secretion were strongly inhibited by antibody directed to CD23. These results clearly show that sCD23 released by HLE or cG stimulated the production of inflammatory mediators by monocytes without any costimulatory signal.

View larger version (36K): [in this window] [in a new window]   Figure 2. Effect of sCD23 produced by HLE or cathepsin G on secretion of TNF- by monocytes. A) Blood monocytes from healthy donors were incubated with supernatant from HLE-treated Daudi cells transfected with cDNA encoding CD23 (CD23+), mock transfected (CD23-), or LPS (1 ng/ml) as indicated. The specificity of CD23 stimulation was evaluated in the presence of a CD23 mAb (+mAb25). TNF- concentrations were measured in culture supernatants using specific ELISA. Data are the mean ± SD of triplicate determinations obtained from 3 distinct experiments. B) Monocytes were incubated with supernatant from HLE, cathepsin G-treated CD23+, or CD23- Daudi cells as indicated. TNF- concentrations were measured and data are the mean ± SD of triplicate determinations obtained from two distinct experiments.

CONCLUSION

In the present report, we show a strong sensitivity of surface CD23 to PMN-derived serine proteases compared with other membrane molecules on B cells. Soluble fragments released by these activities were able to activate resting monocytes without any costimulatory signal as demonstrated by up-regulation of membrane CD14 expression, ROS production, and induction of TNF- secretion. Such proinflammatory effects were similar to those observed with 25 kDa recombinant soluble CD23. Taken together, these data confirm the proinflammatory effects of sCD23 and point out the critical involvement of PMN-derived proteases in their production.

The activities responsible for mCD23 cleavage in our coculture experiments remain to be clarified. The HLE-specific inhibitor partially reversed the diminution of mCD23 (by 65%). We have verified that this inhibitor inactivated purified HLE but not cG, indicating that this latter protease could account for the decrease of mCD23 observed in the presence of the inhibitor. Although we focused here on HLE and cG, we do not exclude the involvement of other PMN-associated activities like proteinase 3. Nevertheless, activities involved in this process must be serine proteases since inhibitors specific to this class completely abolished the cleavage of mCD23 in our coculture conditions. Although metalloproteases have been shown to cleave CD23, we think that proteases of this family account for the release of sCD23 in physiological situations. This study points out the key role of serine proteases rather than metalloproteases in the generation of sCD23 in an inflammatory context.

In inflammatory sites, many activated cells (including monocytes and eosinophils) can express mCD23. A striking correlation has been shown between mCD23 expression on neutrophils and rheumatoid arthritis, in which high levels of sCD23 are detected. The coexistence in inflammatory foci of CD23-bearing cells and PMN-derived serine proteases could explain the large quantity of sCD23 fragments often correlated to inflammatory pathologies.

It is now obvious that proteases from PMN play a major role in tissue injuries in many inflammatory diseases such as rheumatoid arthritis, emphysema, or asthma, but other targets of the extracellular matrix could explain their roles in these disorders. For instance, HLE favors PMN infiltration by cleaving ICAM-1, an integrin required for the firm adherence of PMN to endothelial cells. The present work strongly supports that CD23 cleavage mediated by PMN-derived serine proteases plays a role during the inflammatory response, since we demonstrated that sCD23 released in the presence of these proteases activated monocytes to produce inflammatory mediators.

The sCD23-induced monocyte activation leads to a marked secretion of TNF- that is described to up-regulate expression of receptor for CD23 on monocyte and induce protease degranulation by PMN. Many inhibitors control these proteases in vivo but can be inactivated by oxidation. Thus, diffusible ROS produced by sCD23-activated monocytes might inactivate these inhibitors and enhance proteolytic activities. Consequently, soluble CD23 fragments establish an amplification loop leading to a chronic inflammation (Fig. 3 ). Our model supports results obtained in a collagen-induced murine model of rheumatoid arthritis in which the immunotherapy against CD23 largely improved the disease. This benefit is probably due to the blockade of this loop by preventing the engagement of monocyte receptors by sCD23.

View larger version (36K): [in this window] [in a new window]   Figure 3. Colocalization of CD23-bearing cells and serine protease-producing cells installs and/or maintains an amplification loop leading to an extended production of inflammatory mediators such as TNF- and oxidative entities.

In conclusion, we demonstrate that PMN-derived proteases cleave CD23, releasing proinflammatory soluble fragments. This work strongly supports that colocalization of neutrophils, CD23⁺ cells, and monocytes in inflammatory foci installs an amplifying cascade whereby PMN-derived proteases induce the release of soluble CD23, which activates monocytes to produce mediators of inflammation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0773fje ; to cite this article, use FASEB J. (July 24, 2001) 10.1096/fj.00-0773fje

² Present address: Adult Oncology, Dana Farber Cancer Institute, Boston MA 02115, USA.

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