Cloning and expression of human endothelial-monocyte-activating polypeptide 2 (EMAP-2) and identification of its putative precursor.

Endothelial-monocyte-activating polypeptide 2 (EMAP-2) modulates a range of properties of endothelial cells, monocytes and neutrophils in vitro, and induces an acute inflammatory reaction and tumour regression in vivo. We generated the full-length human cDNA sequences of EMAP-2 and its putative precursor pro-EMAP-2 as PCR products. These were cloned into the pCR3 vector and subcloned into pGEX-2T for expression as fusion products with glutathione-S-transferase (GST). Recombinant EMAP-2 (rEMAP-2) was isolated by thrombin cleavage of the fusion protein, followed by affinity chromatography. rEMAP-2 retained biological activity, which was blocked by polyclonal antibodies raised against GST-EMAP-2. By Western blotting, a 34-kDa product corresponding to the predicted precursor proEMAP-2 was detected in lysates of the U937 monocytic cell line, while supernatants contained higher levels of the mature 22-kDa molecule.

Endothelial-monocyte-activating polypeptide II.

Endothelial-monocyte-activating polypeptide II (EMAP II) was initially identified as a product of murine methylcholanthrene A-induced fibrosarcoma cells. The deduced mRNA sequence indicates that EMAP II is synthesised as a 34 kDa precursor molecule (proEMAP) and enzymatically cleaved to produce a biologically active 22 kDa mature polypeptide, which has been isolated and characterised. It modulates a range of properties of endothelial cells, monocytes, and neutrophils in vitro, and induces an acute inflammatory reaction and tumour regression in vivo. Recent evidence suggests that EMAP II can induce apoptosis in endothelial cells, and as such may act in an anti-angiogenic role. The question arises whether EMAP II is primarily a pro-inflammatory cytokine or a novel mediator of programmed cell death.

Comparison of retroviral p15E-related factors and interferon alpha in head and neck cancer.

Head and neck squamous cell carcinomas (HNscc) produce low-molecular-mass factors (low-M(r) factors, M(r) < or = 25,000), which are antigenically related to the immunosuppressive retroviral transmembrane envelope protein p15E. These P15E-related tumour factors are thought to be responsible for some immunological impairments found in these patients (particularly the defective monocyte chemotaxis). A sequential and functional homology has been reported to exist between a bioactive fragment of interferon alpha (IFN alpha) and the putative immunosuppressive region of retroviral p15E (CKS-17). In this study we investigated (a) a possible functional and structural relationship between p15E and IFN alpha, and (b) the presence of and the relationship between p15E-related low-M(r) factors and IFN alpha in HNscc patients. We report the following results. (a) Recombinant human (rhu) IFN alpha was able to inhibit monocyte chemotaxis. (b) The anti-p15E antibodies crossreacted with rhuIFN alpha in a dot-blot technique; however, the anti-IFN alpha antibodies did not crossreact with disrupted murine leukaemia virus (p15E source). (c) Low-M(r) factors (n = 8-11) prepared from the sera of HNscc patients, which inhibit the monocyte chemotactic responsiveness, could be adsorbed by the anti-p15E antibodies as well as by the anti-IFN alpha antibodies. However, the abilities of the factors to adsorb to the two categories of antibodies (namely, anti-p15E and anti-IFN alpha) did not correlate. (d) Immunohistochemically we found IFN alpha-related epitopes, in almost all HNscc specimens studied (17/18), in locations distinctive from those of p15E-related factors. The anti-IFN alpha antibodies used in this study mainly reacted with basal epithelial cells close to the basal membrane, the prickle and granular cells of the squamous cell carcinomas. The anti-p15E antibodies mainly reacted with corneal layers, the granular and prickle cells, and did not react with basal epithelial cells. Our findings suggest that the immunosuppressive factors produced by HNscc cells are heterogeneous and p15E- and/or IFN alpha-related.

Retroviral p15E-related serum factors and recurrence of head and neck cancer.

Surgical removal of head and neck squamous cell carcinoma (HNSCC) restores the defective monocyte polarization found in patients with HNSCC. Since HNSCC contain p15E-like low molecular weight factors < 25 kD (LMWF) capable of suppressing N-formyl-methionyl-leucylphenylalanine (fMLP)-induced monocyte polarization, it is likely that HNSCC removal eradicates the production site of p15E-like factors. This report describes a prospective follow-up study on the levels of bioactive p15E-like serum factors for a period of 2 years in nine patients with HNSCC who had no recurrence and 11 patients with HNSCC who showed residual or recurrent disease after treatment. In the group of patients without recurrent disease p15E-like bioactivity gradually decreased and eventually became negative. In patients with recurrent/residual disease p15E-like bioactivity remained high or even became positive before or at the time of diagnosing tumour recurrence. This study strongly supports the concept that HNSCC tumours are the production site of p15E-like immuno-suppressive factors and indicates that serum p15E-like factors may be used for future studies on early serum markers for recurrent/residual disease developing in the first year after treatment.

Depressed monocyte polarization and clustering of dendritic cells in patients with head and neck cancer: in vitro restoration of this immunosuppression by thymic hormones.

The in vitro restoring effects of a thymic hormone preparation, TP-1, on defective monocyte and dendritic cell function in patients with head and neck squamous cell carcinoma (HNSCC) have been examined. The N-formylmethionyl-leucyl-phenylalanine (fMLF)-induced polarization of monocytes isolated from the peripheral blood was significantly lower (a mean of 19%) than the polarization of monocytes isolated from healthy controls (a mean of 33%). After the in vitro addition of TP-1 this defective polarization was improved to the normal value of 33% polarized monocytes. The capability of dendritic cells prepared from the blood to form cellular clusters with allogeneic cells was impaired in 26/44 patients. In vitro addition of TP-1 again had restoring effects. The original defective dendritic cell clustering of 97 clusters/six microscopic fields (mean) was improved to a value of 121 clusters. The defects in monocyte polarization and clustering of dendritic cells could be ascribed to the presence in serum of a tumor-derived low-molecular-mass factor low-M(r) factor; < 25 kDa) sharing structural homology with p15E, the capsular protein of murine and feline leukemogenic retroviruses. The incubation of low-M(r) factor from the serum of HNSCC patients with healthy donor monocytes resulted in a significantly higher inhibition of fMLF-induced monocyte polarization than did incubation with control low-M(r) factor (a mean of 42 versus 16% inhibition). This suppressive effect of patient low-M(r) factor was abrogated with a mixture of two monoclonal antibodies against p15E as well as with TP-1. The observations here reported on the in vitro effects of TP-1 on depressed monocyte and dendritic cell function in HNSCC have provided one of the rationales for a TP-1 therapeutic pilot trial recently started in HNSCC patients.