Coexistence of a chemotactic factor and a retroviral P15E-related chemotaxis inhibitor in human tumor cell culture supernatants

Two sets of seemingly contradictory evidence have been reported concerning the effects of tumor cell products on the regulation of monocyte migration in vitro and presumably the extravasation of macrophages into tumors in vivo. The present study was designed to explore the relationship between chemotactic and anti-chemotactic products related to tumor cells: a tumor-derived chemotactic factor (TDCF) and retroviral P15E-related inhibitor(s) of chemotaxis. Culture supernatants of the human 8387 sarcoma and SW626 ovarian carcinoma were depleted of P15E-related antigens with immobilized anti-P15E monoclonal antibodies. This treatment produced a significant and consistent increase of the polarizing and chemotactic activity in the tumor cell supernatants. The material eluted from Sepharose-bound anti-P15E antibodies was devoid of chemotactic and polarizing activity and suppressed the polarization and migration of monocytes in response to chemoattractants. These results demonstrate the coexistence in culture supernatants of two human tumor cell lines of factors with opposite influences on monocyte chemotaxis. The data suggest that the entry of monocytes into neoplastic tissue may be regulated by the interplay of chemotactic and anti-chemotactic principals produced by tumor cells.     

Chemotactic factor and P15E-related chemotaxis inhibitor in human melanoma cell lines with different macrophage content and tumorigenicity in nude mice

The present study was designed to characterize the production of chemoattractants by human melanoma lines with high (M4Be, M3Da, NTerDa) or low tumorigenic (Doc8, M1Do) potential when heterotransplanted in nude mice. Supernatants from the Doc8 and M1Do cell lines were strongly chemotactic in vitro for mononuclear phagocytes. Chemotactic activity was destroyed by proteolytic enzymes, and upon gel filtration on Sephadex G75, it eluted in the cytochrome c region corresponding to an apparent m.w. of 12,000. Upon chromatofocusing, the Sephadex-separated tumor-derived chemotactic factor (TDCF) showed an isoelectric point of 5.5 to 6. Cell lines with high tumorigenic potential contained low or no detectable chemotactic activity. When culture supernatants of cell lines with modest (M3Da) or no (M4Be) chemotactic activity were exposed to immobilized monoclonal antibodies directed against the retroviral transmembrane protein P15E, appreciable chemotactic activity was detectable (M4Be) or preexisting levels increased (M3Da). The material eluted from Sepharose-bound anti-P15E antibodies inhibited the migration of monocytes in response to chemoattractants. These findings demonstrate the coexistence in some human melanoma cell line supernatants of factors (TDCF and P15E-related inhibitor) with opposite influence on monocyte chemotaxis. That tumor cell products play a pivotal role in regulating the extravasation of monocytes into neoplastic tissues is suggested by the close correlation observed between macrophage levels in melanomas grown in nude mice and levels of chemotactic activity detectable in culture supernatants.     

IL-1 stimulates IL-6 production in endothelial cells

Leukocytes and vascular cells interact closely in inflammation and immunity and lymphokines are important mediators of this interaction. The present study was designed to define the possible role of IL-6 as a communication signal between vascular and immunocompetent cells. IL-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line in the supernatants of human endothelial cells (HEC). HEC released appreciable levels of HGF activity in the absence of deliberate stimulation. In vitro exposure to recombinant IL-1 beta markedly increased (usually 10 to 15-fold) HGF production by HEC. Optimal stimulation was observed with 0.1 to 50 U/ml for 4 to 20 h of incubation. Human and murine rIL-1 alpha stimulated HGF production in HEC. Anti-IL-6 antibodies inhibited the HGF activity of the HEC supernatants, thus confirming, together with the cytokine specificity of the assay, the nature of HEC-produced cytokine. IL-1-treated HEC expressed high levels of IL-6 mRNA as detected by Northern blot analysis. Inasmuch as IL-1 elicits a complex series of changes in HEC, it was important to assess whether IL-6, produced after exposure to IL-1, modified HEC function. Natural or rIL-6 did not affect the functional status of HEC as assessed by proliferative capacity, production of procoagulant activity and prostacyclin, ability to induce adhesion of polymorphonuclear leukocytes. The capacity to produce IL-6 may represent an important mechanism by which endothelial cells participate in inflammatory and immune reactions.     

Antiinflammatory proteins associated with human and murine neoplasms

The immune mechanisms by which a host recognizes and destroys a growing tumor are undoubtedly complex and, as yet, incompletely understood. It is apparent, however, that mononuclear phagocytes play an important role in the defense against neoplastic disease and that the ability of monocytes and macrophages to accumulate at and within a growing tumor is a strict requirement for them to effect that role. Studies from our laboratory as well as those of other investigators have demonstrated that patients with a variety of neoplastic diseases have a specific defect in monocyte chemotactic responsiveness and that this defect is associated with the presence of the tumor. Furthermore, we and others have shown that a similar defect occurs in tumor-bearing rodents, thus allowing model systems to be developed for the study of the mechanisms involved. We have demonstrated that transplanted, spontaneous or carcinogen-induced murine tumors produce low molecular weight proteins which inhibit the accumulation of macrophages to inflammatory foci and that a significant portion, if not all, of these proteins are physicochemically and antigenically related to the retroviral envelope protein p15E. We have shown that p15E itself can inhibit the inflammatory accumulation of macrophages in normal mice. Studies on a wide variety of cancer patients have revealed that the fluids of such patients contain proteins which inhibit the responses of normal monocytes to various chemotaxins and, as in tumor-bearing mice, that these antiinflammatory proteins are antigenically related to retroviral p15E. Recent studies have demonstrated that human tumor cells can simultaneously release factors which are chemotactic for monocytes with those which are p15E-related inhibitors of chemotactic responsiveness, suggesting that the mononuclear phagocyte response to a growing tumor may be, in part, dictated by the balance obtained between various proteins produced by that tumor. The isolation and characterization of endogenous retroviral sequences within the human genome and the observation that the envelope genes of these endogenous sequences are partially homologous to p15E provide potential candidates for the p15E-related inhibitors of chemotactic responses which have been identified from human cancer cells and fluids. Studies now under way in a number of laboratories should provide more definitive answers regarding the nature and source of these p15E-related inhibitors.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

Head and neck squamous cell carcinomas (HNscc) produce low-molecular-mass factors (low-M _r factors,M _r25,000), 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 (IFN) 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, and (b) the presence of and the relationship between p15E-related low-M _r factors and IFN in HNscc patients. We report the following results. (a) Recombinant human (rhu) IFN was able to inhibit monocyte chemotaxis. (b) The anti-p15E antibodies crossreacted with rhuIFN in a dot-blot technique, however, the anti-IFN 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 antibodies. However, the abilities of the factors to adsorb to the two categories of antibodies (namely, anti-p15E and anti-IFN) did not correlate. (d) Immunohistochemically we found IFN-related epitopes, in almost all HNscc specimens studied (17/18), in locations distinctive from those of p15E-related factors. The anti-IFN 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-related.     

Regulation of IL-6 expression by oncostatin M

Endothelial cells produce immunomodulatory cytokines in response to soluble mediators of inflammatory/immune reactions. We have previously demonstrated that the leukocyte-derived cytokine, oncostatin M (Onco M) can alter endothelial cell morphology, regeneration, and fibrinolytic activity in vitro. Here we demonstrate that Onco M stimulates the production of the pleiotropic cytokine, IL-6, in cultured human endothelial cells (HEC) in a time- and dose-dependent manner. Specific antibodies to IL-6 neutralize the growth-inhibitory activity for human breast carcinoma cells that is secreted by HEC in response to Onco M treatment. Specific immunoassays indicate greater than 10-fold increases in the IL-6 content of culture supernatants as early as 6 h post-treatment with Onco M (ED50 = 17 pM). This stimulation of IL-6 production by Onco M is associated with a sevenfold increase in intracellular levels of IL-6 mRNA. IL-1 alpha and TNF-alpha are also potent inducers of IL-6 production in these cells, the order of potency being IL-1 alpha greater than Onco M greater than TNF-alpha. TNF-alpha, but not IL-1 alpha, synergizes with Onco M to augment IL-6 production in HEC. HEC are 10 to 20 times more responsive to Onco M than are other nonendothelial cell types. In addition, HEC express 10 to 20 times greater numbers of high affinity cell-surface receptors for Onco M than do other nonendothelial cell types. Based on these findings, we propose that Onco M may represent a new immunomodulator regulating cytokine-induced gene products in endothelial cells.     

Oncostatin M induces tyrosine phosphorylation in endothelial cells and activation of p62yes tyrosine kinase.

Oncostatin M is a polypeptide cytokine produced by activated and transformed T lymphocytes that has diverse biologic effects, including growth inhibition of tumor cells and induction of IL-6 expression in cultured human endothelial cells (HEC). HEC are highly responsive to oncostatin M and express high levels of oncostatin M receptors relative to other cell types. Oncostatin M has previously been found to bind a specific receptor of 150 to 160 kDa. We have found through the use of anti-phosphotyrosine immunoblotting that oncostatin M induces tyrosine phosphorylation in HEC. Anti-phosphotyrosine antibodies specifically immunoprecipitated labeled oncostatin M cross-linked to its receptor, demonstrating that the oncostatin M receptor is either directly phosphorylated on tyrosine after ligand binding or is tightly associated with a phosphotyrosyl protein in these cells. The tyrosine kinase inhibitor herbimycin A blocked the induction of IL-6 by oncostatin M in HEC. In addition, immune complex kinase assays showed that oncostatin M markedly increased the activity of the p62yes tyrosine kinase with a small increase in p59fyn but no increase in p56lyn tyrosine kinase activity in HEC. We conclude that oncostatin M utilizes a tyrosine phosphorylation signal transduction pathway in HEC involving the activation of the p62yes tyrosine kinase, and that this tyrosine phosphorylation pathway leads to the induction of IL-6 expression.     

The pharmacologic regulation of interleukin-1 production: the role of prostaglandins

The role of prostaglandins in the regulation of lipopolysaccharide (LPS)-induced interleukin-1 (IL-1) production by murine C3H/HeN resident peritoneal macrophages was studied. IL-1 production was initially studied in the presence of piroxicam and indomethacin, both inhibitors of prostaglandin biosynthesis. IL-1 was assayed using the IL-1-dependent proliferative response of C3H/HeJ thymocytes. LPS stimulation resulted in 15 to 20 ng/ml of prostaglandin E2 (PGE2) produced in the first hour of culture. IL-1-containing supernatants from drug-treated macrophages at dilutions of up to 1:32 resulted in enhanced thymocyte proliferation compared to control, non-drug-treated cultures and contained less than 2 ng/ml of PGE2. Similar enhancement of proliferation could be obtained by incubating non-drug-treated supernatants with monoclonal anti-PGE2 but not anti-thromboxane B2 (TxB2) antibody. Further dilutions of the drug-treated supernatants gave thymocyte proliferation responses which were indistinguishable from control cultures and, correspondingly, had identical values for IL-1 production. The absence of an effect on IL-1 production was confirmed by quantitation of intracellular IL-1 alpha using goat anti-IL-1 alpha antibody and by quantitation of supernatant IL-1 receptor competition assay. Exogenous PGE2, in the concentration range produced in macrophage supernatants (10-20 ng/ml), directly inhibited IL-1-stimulated thymocyte proliferation. Finally, when macrophages were stimulated with LPS for 24 hr in the presence of added PGE2, thymocyte proliferation was inhibited at the lowest supernatant dilutions, but as the IL-1-containing supernatants were diluted out, the assay curves were indistinguishable from non-PGE2-treated control. Thus, in this system, PGE2 has no effect on IL-1 synthesis, but rather has a direct inhibitory effect on thymocyte proliferation. Nonsteroidal anti-inflammatory drugs are not stimulating IL-1 production but are, in fact, relieving inhibition of the thymocyte IL-1 assay caused by the presence of prostaglandins.     

Differential expression of CC chemokines and the CCR5 receptor in the pancreas is associated with progression to type I diabetes

We investigated the biological role of CC chemokines in the Th1-mediated pathogenesis of spontaneous type I diabetes in nonobese diabetic (NOD) mice. Whereas an elevated ratio of macrophage inflammatory protein-1alpha (MIP-1alpha):MIP-1beta in the pancreas correlated with destructive insulitis and progression to diabetes in NOD mice, a decreased intrapancreatic MIP-1alpha:MIP-1beta ratio was observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment, which prevents diabetes in NOD mice by polarizing intraislet Th2 responses, decreased CCR5 expression in islets and potentiated a high ratio of MIP-1beta and monocyte chemotactic protein-1 (MCP-1): MIP-1alpha in the pancreas. Furthermore, NOD.MIP-1alpha-/- mice exhibited reduced destructive insulitis and were protected from diabetes. Neutralization of MIP-1alpha with specific Abs following transfer of diabetogenic T cells delayed the onset of diabetes in NOD.Scid recipients. These studies illustrate that the temporal expression of certain CC chemokines, particularly MIP-1alpha, and the CCR5 chemokine receptor in the pancreas is associated with the development of insulitis and spontaneous type I diabetes.     

A synthetic peptide homologous to the envelope proteins of retroviruses inhibits monocyte-mediated killing by inactivating interleukin 1

The synthetic peptide CKS-17 has homology to a highly conserved region of the immunosuppressive retroviral envelope protein P15E, to envelope proteins of HTLV I, II, III, and to that encoded by an endogeneous C-type human retroviral DNA. CKS-17 inhibits the immune response of lymphocytes and the respiratory burst of human monocytes. Because P15E-related antigens are present in human malignant cell lines and cancerous effusions, we sought to determine the effect of CKS-17 on monocyte-mediated tumor cell lysis. Lysis of A375 tumor cells by lymphokine or lipopolysaccharide-activated human monocytes was inhibited by 10 microM CKS-17 (control, 79%; CKS-17-treated, 19%). Another synthesized peptide, CS-2, which has partial homology to CKS-17, failed to block monocyte-mediated killing. Thus, the inhibition by CKS-17 appeared to be specific. Because interleukin 1 (IL-1) is a cytocidal factor produced by activated monocytes, we also investigated the effect of CKS-17 on IL-1 production by monocytes and on direct IL-1-mediated cytotoxicity. CKS-17 did not block production or secretion of IL-1 by lipopolysaccharide- or interferon-gamma-activated monocytes. However, the direct cytocidal activity of both recombinant IL-1 alpha and IL-1 beta against A375 tumor cells was blocked by CKS-17. The cytotoxic activity of IL-1 was inhibited by CKS-17 if (a) IL-1 was preincubated with CKS-17 for 1 hr at 37 degrees C or (b) the A375 cells were incubated with CKS-17 for 1 hr prior to the addition of IL-1. CKS-17 also blocked IL-1-induced proliferation of murine thymocytes, the D10 T cell line, and an IL-1-responsive astrocytoma cell line. These data suggest that CKS-17 may be a potent inhibitor of IL-1.     

IL-7 up-regulates the expression of IL-8 from resting and stimulated human blood monocytes.

Blood monocytes are important cellular sources of a vast array of bioactive substances, including regulatory and chemotactic cytokines. The regulation of these cytokines is of critical importance to the expression of acute and chronic inflammatory responses. IL-7, a T and B cell-activating cytokine, has recently been shown to have stimulatory effects on the expression of several monocyte-derived proinflammatory cytokines. We now describe the induction of IL-8 mRNA and extracellular protein from human blood monocytes by IL-7. The up-regulation of IL-8 mRNA by IL-7 was not altered by concomitant treatment with cycloheximide, suggesting that the direct stimulatory effects of IL-7 were not dependent upon de novo protein synthesis. In addition, IL-7 significantly potentiated the production of IL-8 from LPS-, TNF-, and IL-1-treated peripheral blood monocytes. Our findings suggest that IL-7 may play a critical role in the modulation of macrophage-derived cytokine expression and may function in vivo as an important proinflammatory cytokine.     

Generation of lipid neutrophil chemoattractant by irradiated bovine aortic endothelial cells

Radiation injury to blood vessels is associated with an acute inflammatory process. We investigated the capacity of cultured bovine aortic endothelial cells (BAEC) to produce chemotactic factors after radiation injury. BAEC in serum-free media were irradiated with a cobalt-60 Gammacell 220 and the cell supernatants were assayed for chemotactic activity for human neutrophils in a Boyden chamber. There was a rapid release of chemotactic activity into the BAEC supernatants which was dependent both on the dose of radiation (5 to 40 Gy) and the time between irradiation and sample collection. In contrast, isolation of BAEC lysates by freeze-thawing was not associated with the presence of similar chemotactic activity. The chemotactic activity released from the irradiated BAEC was not destroyed by boiling nor by treatment with trypsin. The release of the chemotactic activity was, however, inhibited by the addition of a lipoxygenase inhibitor but not by the addition of a cyclooxygenase inhibitor before the irradiation. The chemotactic activity was recovered from the cell supernatants in the lipid phase after extraction with chloroform/methanol. Furthermore, the chloroform/methanol extracts co-eluted with authentic leukotriene B4 when the BAEC were prelabeled with [14C] arachidonic acid. However, we were unable to detect endogenous leukotriene B4 with RIA. Instead, the only detectable endogenous lipid present in the supernatants was 13-hydroxyoctadecadienoic acid which is derived from linoleic acid via the lipoxygenase pathway. 13-Hydroxyoctadecadienoic acid, however, had no chemotactic activity. These findings suggest that endothelial cells rapidly release a chemotactic agent after irradiation, the release of which is associated with a lipoxygenase pathway. The release of this chemotactic activity may account in part for the acute inflammatory response that is observed after ionizing irradiation.     

IL-32γ induces chemotaxis of activated T cells via dendritic cell-derived CCL5

Interleukin (IL)-32 has been associated with a variety of inflammatory diseases including rheumatoid arthritis, vasculitis and Crohn’s disease. We have previously reported that IL-32γ, the IL-32 isoform with the highest biological activity, could act as an immune modulator through regulation of dendritic cell (DC) functions in immune responses. Cell locomotion is crucial for induction of an effective immune response. In this study, we investigated the effect and underlying mechanisms of IL-32γ on recruitment of T cells. IL-32γ upregulated the expression of several chemokines including CCL2, CCL4, and CCL5 in the DCs. In particular, IL-32γ significantly increased CCL5 expression in a dose-dependent manner. Treatment with JNK and NF-κB inhibitors suppressed IL-32γ-induced CCL5 expression in DCs, indicating that IL-32γ induced CCL5 production through the JNK and NF-κB pathways. Furthermore, supernatants from IL-32γ-treated DCs showed chemotactic activities controlling migration of activated CD4⁺ and CD8⁺ T cells, and these activities were suppressed by addition of neutralizing anti-CCL5 antibody. These results show that IL-32γ effectively promotes migration of activated T cells via CCL5 production in DCs. The chemotactic potential of IL-32γ may explain the pro-inflammatory effects of IL-32 and the pathologic role of IL-32 in immune disorders such as rheumatoid arthritis.     

The role of neutrophil membrane glycoprotein 150 (Gp-150) in neutrophil-mediated endothelial cell injury in vitro

In this study we examined the importance of neutrophil adherence in neutrophil-mediated endothelial cell injury. Phorbol myristate acetate (PMA)-activated neutrophils from a patient with a congenital defect in neutrophil adherence (Gp-150 deficiency) and PMA-activated normal neutrophils pretreated with monoclonal antibody (MoAb) 60.3 were used. Both Gp-150-deficient and MoAb 60.3-treated normal neutrophils failed to adhere to cultured human umbilical vein endothelial cell (HEC) monolayers when activated by PMA (adherence less than 10% with patient and MoAb 60.3-treated cells compared with 53 +/- 3% with normal cells). The addition of PMA-activated normal neutrophils to 51Cr-labeled HEC monolayers failed to induce significant 51Cr release but did produce marked HEC detachment (percentage of detachment 50 +/- 3 at 6 hr). In marked contrast, PMA-activated Gp-150-deficient neutrophils failed to induce significant HEC detachment (percentage of detachment zero (0) at 6 hr). Moreover, the addition of MoAb 60.3 to normal neutrophils inhibited neutrophil-mediated HEC detachment in a time- and dose-dependent fashion. Non-lytic HEC detachment was determined to be largely oxygen radical independent, because PMA-activated chronic granulomatous disease neutrophils and PMA-activated normal neutrophils produced similar disruption of HEC monolayers. Soybean trypsin inhibitor, a chloromethylketone elastase inhibitor, and autologous serum all failed to inhibit neutrophil-mediated HEC detachment. From these studies there is no evidence that nonlytic HEC detachment by PMA-activated neutrophils is mediated by the neutrophil-derived proteases, elastase and cathepsin G. Neutrophil-mediated HEC detachment also required intact neutrophils, because postsecretory medium from PMA-activated normal neutrophils and a suspension of frozen-thawed PMA-activated normal neutrophils were without effect. These in vitro studies indicate that the neutrophil cell surface glycoprotein Gp-150 is required for nonlytic HEC detachment by intact PMA-activated neutrophils.     

Plasminogen activator inhibitor-1 supports IL-8-mediated neutrophil transendothelial migration by inhibition of the constitutive shedding of endothelial IL-8/heparan sulfate/syndecan-1 complexes

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.     

Contribution of IL-1 beta and TNF-alpha to the initiation of the peripheral lung response to atmospheric particulates (PM10)

Alveolar macrophages (AM) play a key role in clearing atmospheric particulates from the lung surface and stimulating epithelial cells to produce proinflammatory mediators. The present study examines the role of "acute response" cytokines TNF-alpha and IL-1 beta released by AM exposed to ambient particulate matter with a diameter of <10 microm (PM(10)) in amplifying the proinflammatory mediator expression by A549 cells and human bronchial epithelial cells (HBEC). The results showed that supernatants from human AM incubated 24 h with PM(10) (100 microg/ml) contained more TNF-alpha, IL-1 beta, granulocyte-macrophage colony stimulating factor, IL-6, and IL-8 than nonexposed AM supernatants. The 3-h treatment of A549 cells with PM(10)-exposed AM supernatants increased TNF-alpha, IL-1 beta, IL-8, regulated on activation normal T-cells expressed and secreted (RANTES), and leukemia inhibitory factor mRNA compared with the treatment with nonexposed AM supernatants and, compared with untreated A549 cells, additionally increased ICAM-1 and monocyte chemotactic protein-1 mRNA. Preincubating PM(10)-exposed AM supernatants with anti-IL-1 beta antibodies reduced all the above mediators as well as VEGF mRNA expression (P < 0.05), while anti-TNF-alpha antibodies were less effective (P > 0.05), and the combination of the two antibodies most effective. When HBEC were treated similarly, anti-TNF-alpha antibodies had the greatest effect. In A549 cells PM(10)-exposed AM supernatants increased NF-kappa B, activator protein (AP)-1 and specificity protein 1 binding, while anti-TNF-alpha and anti-IL-1 beta antibodies reduced NF-kappa B and AP-1 binding. We conclude that AM-derived TNF-alpha and IL-1 beta provide a major stimulus for the production of proinflammatory mediators by lung epithelial cells and that their relative importance may depend on the type of epithelial cell target.     

富含二硫键的膜蛋白p185胞外区在大肠杆菌中的可溶性表达和性质鉴定

Her2/c-erbB-2基因（其产物为膜蛋白p185）是表皮生长因子受体（EGFR）基因家族的一员,在约30％的乳腺癌中发现了其过量表达。为了鉴定抗p185单克隆抗体的抗原表位并进一步研究它们的相互作用,采用PCR的方法从含Her2/c_erbB_2基因的pBabe/erbB_2质粒中扩增了p185胞外区的富含二硫键的第一、二结构域和第四个结构域。产物克隆到pGEX/4T-1载体后,转化大肠杆菌Origami B（DE3）pLysS菌株,用低浓度IPTG进行低温过夜诱导后将菌体压力破碎,SDS-PAGE检测表达上清,得到了可溶性表达的融合有GST的目的蛋白。经ELISA、Western blot等方法鉴定,可溶性表达产物具有完全的抗体结合活性,且当用凝血酶把GST切掉后该活性仍然保留。P185胞外区融合蛋白的成功表达将为二硫键富含类蛋白的表达提供参考;并为将来具有肿瘤细胞生长抑制活性的抗p185单克隆抗体的抗原表位鉴定,以及为EGFR家族受体的结构和功能关系的研究打下基础。