Chemokines are secreted by monocytes following OK-432 (lyophilized Streptococcus pyogenes) stimulation

Abstract

Background: OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied in vitro the role of mononuclear phagocytes (MNPs), including purified monocytes (MOs), in the immune response to OK-432. MIP-1alpha/beta and MCP-1 secretions were assessed in whole blood (WB), peripheral blood mononuclear cells (PBMCs) and purified MOs, after in vitro stimulation with OK-432 with or without adherence for 24 hours.

Results: OK-432 stimulated MNPs to secrete MCP-1 and MIP-1alpha/beta in healthy individuals and in head and neck squamous cell carcinoma (HNSCC) patients, except for OK-432 stimulation of WB giving a minimal MIP-1alpha/beta response. Upon culture on low-attachment wells, a spontaneous chemokine secretion was observed, with an unchanged secretion following OK-432 stimulation. Inhibition of Syk kinase and/or PI-3 kinase did not significantly change the chemokine response to OK-432, except for MIP-1alpha production being increased upon Syk inhibitor addition and an increased MCP-1 response upon addition of both inhibitors. Adhesion may possibly involve beta1 and/or beta3 integrins, not beta2, whereas beta(1-3) integrins may act as co-stimulatory receptors for OK-432. Based on direct blockage of CD36 or CD18 by antibodies, MCP-1 production may be mediated by CD18 while MIP-1beta and MCP-1 production may occur upon binding to CD36.

Conclusion: Adherent human MOs produce MCP-1 and MIP-1alpha/beta upon stimulation with OK-432. CD36 modulates MIP-1beta and MCP-1 response. Thus, to some extent OK-432 acts as a substance whereby only MOs adhered to surfaces secrete MCP-1 and MIP-1alpha/beta, in part explaining why OK-432 is suited as a biological response modifying drug.

Figure 1

In vitro chemokine production in unstimulated WB, PBMCs or MOs. Human WB, PBMCs or MOs were plated in parallel on regular (R) or low attachment (L) plates and left unstimulated for 24 h (A). Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP-1). WB (n = 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6).

Figure 2

OK-432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients. Human WB, PBMCs or MOs were stimulated with media or OK-432 (0.01 KE/ml) for 24 h (A). MOs isolated from four HNSCC patients were stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12); MO (n = 27); B: HNSCC (n = 4).

Figure 3

OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and correlates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin. MCP-1 gene expression was assessed in MOs stimulated with OK-432 for 24 h from three randomly selected healthy donors (A) or two HNSCC patients on two separate days (C). In separate experiments, MOs, stimulated with piceatannol (120 μM) (B) or wortmannin (100 nM) (D) for 30 min followed by stimulation with OK-432 for an additional 24 h, were analyzed by ELISA or real-time PCR with respect to MCP-1 production and gene expression, respectively. A, B, and D: n = 3; C: n = 2.

Figure 4

Chemokine production follows OK-432, LTA or LPS in vitro stimulation of WB. Human WB was stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP-1). Control, OK-432, LPS: n = 8; LTA: n = 4.

Figure 5

OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432, LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n = 10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n = 9; LPS: n = 16).

Figure 6

OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs. WB, PBMCs or MOs were stimulated with OK-432 (0.01 KE/ml) or left unstimulated on regular (R) or low-attachment (L) plates for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8; PBMC: n = 8; MO: n = 6.

Figure 7

MO-depleted PBMCs secrete less chemokines than PBMCs upon OK-432 stimulation. PBMCs, MOs, or PBs (MO-depleted PBMCs) were left unstimulated or stimulated in parallel with OK-432 (0.01 KE/ml) or LPS (1 μg/ml) for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). n = 2.

Figure 8

Syk and PI-3 kinase-independent signaling for OK-432 stimulation of MOs. Piceatannol (1 μM) or diluent control was added to MO cultures for 30 min prior to addition of OK-432 for 24 h (A). LY294002 (50 μM) was added to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50 μM) and piceatannol (30 μM) was added to whole blood and throughout the monocyte isolation procedure (C) and followed by stimulation with OK-432 or left unstimulated for 24 h (D). Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4.

Figure 9

OK-432 promotes Syk or PI-3 kinase phosphorylation in MOs. MOs, isolated by the monocyte negative isolation kit, were incubated either with piceatannol, Pic. (30 μM) or diluent control (A) or LY294002, LY (50 μM) (B) and either left unstimulated or further stimulated with OK-432 for 15 min. Blots shown are representative of two separate experiments performed with pooled MOs obtained from two different blood donors.

Figure 10

Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti-CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incubated either with isotype control or anti-CD36 (2.5 μg/ml) (B) prior to incubation with OK-432 for 24 h. Supernatants were analyzed for shown chemokines by Multiplex (MIP) or ELISA (MCP-1). A: n = 3; B: n = 6. CD18: MCP-1 (NS).