GRO-α Expression Research Articles

Expression and production of the CXC chemokine growth-related oncogene-alpha by human eosinophils.

Eosinophils are seen together with neutrophils at sites of inflammation. However, their roles are not clear. In addition, eosinophils infiltrate tumor tissue in some neoplastic diseases. In this study, we show that large amounts of the neutrophil-activating CXC chemokine growth-related oncogene (GRO)-alpha can be produced by human eosinophils. Eosinophils showed presence of preformed GRO-alpha in the crystalloid-containing specific granules (190 pg/2 x 10(6) cells). During incubation, a strong increase in GRO-alpha gene expression was seen. At a low cell density, addition of TNF-alpha or IL-1 beta increased the production of GRO-alpha in eosinophils, which was not the case at a higher cell density. Eosinophils can produce TNF-alpha themselves, and neutralizing Abs against TNF-alpha significantly inhibited GRO-alpha production. This suggests that autocrine and paracrine effects from TNF-alpha can be important when up-regulating GRO-alpha gene expression. In contrast, IFN-gamma, a prototypic Th1-cytokine, down-regulated expression of GRO-alpha. This may be important during resolution of inflammation but also suggests different roles for eosinophils depending on the inflammatory context. Tumor-infiltrating eosinophils in Hodgkin's disease of the nodular sclerosing type are associated with a poor prognosis. Eosinophils from such tumor tissue showed an abundant expression of GRO-alpha. The GRO-alpha receptor CXCR2 was also detected in tumor tissue, proposing interactions between eosinophils and the tumor. Our findings suggest that eosinophils can promote inflammation through recruitment of CXCR2-bearing cells. In addition, this feature of the eosinophils indicates a role for these cells in the biology of certain tumors.

Interleukin-13 and tumor necrosis factor-β differentially regulate the production of cytokines by cultured human endometrial stromal cells

Objective To evaluate the effects of interleukin (IL)-13, a T-helper (Th)2 cytokine, and tumor necrosis factor (TNF)-β, a Th1 cytokine, on the production of IL-6 family cytokines and chemokines by endometrial stromal cells (ESC). Design The effects of IL-13 and TNF-β, on the production of IL-6, IL-11, leukemia inhibitory factor (LIF), IL-8, growth-regulated oncogene α (GROα), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, T-cell expressed and secreted (RANTES), and eotaxin were investigated. Setting Research laboratory at a medical university. Patient(s) Thirteen endometrial specimens in the late proliferative phase were used. Intervention(s) The ESC were incubated for 24 hours with recombinant human IL-13 and recombinant human TNF-β. Main outcome measure(s) The concentration of IL-6, IL-11, LIF, IL-8, GROα, MCP-1, RANTES, and eotaxin in the culture media was measured using ELISA. Result(s) The increase in levels of IL-6, IL-8, MCP-1, and eotaxin in the culture media of ESC paralleled the addition of increasing amounts of IL-13 and TNF-β, whereas the levels of IL-11 and LIF were decreased with increasing amounts of IL-13, but were increased with increasing amounts of TNF-β. Tumor necrosis factor-β enhanced the production of GROα and RANTES in dose-dependent manner; however, IL-13 did not affect the expression of GROα or RANTES. Conclusion(s) These results suggest that IL-13 and TNF-β secreted in the cyclic endometrial tissue and in the decidua may differentially regulate the production of IL-6 family cytokines and chemokines by ESC. The controlled expression of these cytokines in the endometrium may contribute to the modulation of the immune reaction during the menstrual cycle and in early pregnancy by the regulation of leukocyte trafficking and functions.

Production of growth related oncogene protein-α in human umbilical vein endothelial cells stimulated with soluble interleukin-6 receptor-α: role of signal transducers, janus kinase 2 and mitogen-activated kinase kinase

Growth-related oncogene protein-α (GRO-α) is a member of the C-X-C chemokine family with a wide variety of biological activities. We studied the production of GRO-α by human umbilical vein endothelial cells (HUVEC) in response to the stimulation with soluble form of interleukin-6 receptor α (sIL-6R). sIL-6R stimulated HUVEC to express GRO-α mRNA and secrete GRO-α protein in concentration-and time-dependent manners. The sIL-6R-induced GRO-α expression was inhibited by the pretreatment of the cells with AG490, a janus kinase 2 (JAK2) inhibitor, or with U0126, a MAP kinase-ERK kinase (MEK) inhibitor. sIL-6R also induced the phosphorylation of both Src homology 2-protein tyrosine phosphatase-2 (SHP-2), signal transducer and activator of transcription 3 (STAT3) and MEK. AG490 pretreatment inhibited the MEK phosphorylation but did not affect the STAT3 phosphorylation. We conclude that sIL-6R induces GRO-α expression in HUVEC through the activation of JAK2 and MEK.

Influence of the cellular redox state on NF-kappaB-regulated gene expression.

The redox state has been shown to regulate a variety of biochemical functions including cellular proliferation. Previous studies from our laboratory and others have shown that the binding of many transcription factors to their cognate DNA sequences is sensitive to the redox environment. Therefore, it is likely that redox status serves as an additional regulatory control for the activity of transcription factors and that this may mediate the redox regulation of proliferation. To assess this possibility, the influence of altering the redox state on NF-kappaB-regulated gene expression was studied. A more-reducing environment favored higher levels of expression of gro, an endogenous gene associated with proliferation, when the redox levels were changed either naturally by altering culture density or chemically by treatment with modulators of glutathione synthesis. Furthermore, nuclear runoff studies showed that a more-reducing redox increased transcription of gro. In order to ascertain the singular effect of the redox state on the activity of NF-kappaB, expression of a secreted alkaline phosphatase (SEAP) reporter gene solely under the control of an NF-kappaB response element was measured under varying redox conditions. Changes in the redox state modulated the expression of this reporter system. Taken together, these results suggest the involvement of a redox mechanism regulating signaling events operating through the control of gene expression by transcription factors.

Influence of the cellular redox state on NF-κB-regulated gene expression

The redox state has been shown to regulate a variety of biochemical functions including cellular proliferation. Previous studies from our laboratory and others have shown that the binding of many transcription factors to their cognate DNA sequences is sensitive to the redox environment. Therefore, it is likely that redox status serves as an additional regulatory control for the activity of transcription factors and that this may mediate the redox regulation of proliferation. To assess this possibility, the influence of altering the redox state on NF-κB-regulated gene expression was studied. A more-reducing environment favored higher levels of expression of gro, an endogenous gene associated with proliferation, when the redox levels were changed either naturally by altering culture density or chemically by treatment with modulators of glutathione synthesis. Furthermore, nuclear runoff studies showed that a more-reducing redox increased transcription of gro. In order to ascertain the singular effect of the redox state on the activity of NF-κB, expression of a secreted alkaline phosphatase (SEAP) reporter gene solely under the control of an NF-κB response element was measured under varying redox conditions. Changes in the redox state modulated the expression of this reporter system. Taken together, these results suggest the involvement of a redox mechanism regulating signaling events operating through the control of gene expression by transcription factors. J. Cell. Physiol. 183:45–52, 2000. © 2000 Wiley-Liss, Inc.

Inhibition of pulmonary microvascular chemokine production by human Laktoferrin and Phosphatidylethanolamine

Chemokines, a large family of structurally related chemotactic cytokines, are essential for the migration of leukocytes during inflammatory processes. The release of neutrophil attractant chemokines such as MCP-1, Groα, ENA-78, GCP-2 and NAP-2 as well as fractalkine by lung microvascular endothelial cells (LMVEC) is dramatically increased upon stimulation with LPS. The present study was performed to investigate, whether human Lactoferrin (hLf) or Phospholipase A2-inhibitor Phosphatidylethanolamine (PE) can intervene with the release of these chemokines by LMVEC under LPS stimulation. LMVEC were protreated with hLf for various time intervals prior to stimulation with LPS or with LPS in the presence of hLf. In other experiments, cell impermeable PE, Cyclooxygenase-inhibitors (Col) (indomethacine, acetyl-salicylic acid) and a platelet activating factor (PAF)-antagonist, were added to LPS stimulated LMVEC. Chemokine release was measured by ELISA and detection of chemokine mRNA by means of RT-PCR. HLf added to LMVEC at the time of stimulation did not influence chemokine production. However, when hLf was added prior to LPS stimulation, a significant inhibition of MCP-1 (P < 0.001) and ENA-78 (P < 0.01) but not of Groα production was observed. In order to investigate if LPS induced chemokine production was dependent on PAF, Arachidonic acid (AA) or its metabolites, LMVEC were treated with PE, Col and PAF-antagonist either after or at the time of LPS stimulation. Treatment of LMVEC with PE completely inhibited the LPS induced chemokine production of MCP-1, ENA-78 and Groα in a time and dose dependent fashion. This was still observed, when LMVEC were pretreated with LPS. Col and PAF-antagonist could partly reduce LPS induced chemokine production. Furthermore mRNA expression of ENA-78, Groα, GCP-2 and fractalkine was decreased after LPS stimulation in the presence of PE. Our data demonstrate, that hLf and PE are potent agents to counteract LPS induced chemokine produktion by LMVEC. These findings may have clinical implications in the treatment of acute lung injury during sepsis.

Expression of Chemokines RANTES, MIP-1α and GRO-α Correlates with Inflammation in Acute Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an investigator-initiated disorder that serves as an animal model for the common human demyelinating disease multiple sclerosis. Both diseases are typified by disseminated perivascular and submeningeal cuffs in the central nervous system (CNS). It was shown recently that chemokines are integral to the pathogenesis of EAE. In the present study we analyzed the gene expression of three chemokines, RANTES, MIP-1α and GRO-α, at the onset of acute EAE, and correlated that expression with the intensity of inflammatory changes in the CNS. We showed that all three chemokines are upregulated simultaneously with symptom onset of acute EAE, and that chemokine expression correlates with the intensity of inflammation in the CNS. This consistent relationship supports the hypothesis that chemokines are relevant to leukocyte accumulation in CNS parenchyma.

Differential Expression of GRO-α and IL-8 mRNA Psoriasis: A Model for Neutrophil Migration and Accumulation In Vivo

Dense focal accumulation of neutrophils in the upper epidermis is a hallmark of psoriasis. Because the signals for neutrophil diapedesis and migration in vivo are not fully understood, psoriatic lesions with pronounced migration of neutrophils may serve as an important model for studying neutrophil chemotaxis. In this study, we present evidence for differential expression of the neutrophil chemotactic cytokines growth-related oncogene alpha, interleukin-8, and ENA-78 (epithelial cell derived and neutrophil-activating properties, 78 amino acids) in psoriatic lesions. In situ hybridization and immunohistochemistry of serial sections were employed to identify and microanatomically localize the cells producing these chemokines. High levels of focal interleukin-8 message were found to be expressed in the upper epidermis by keratinocytes and, most importantly, neutrophils themselves. Growth-related oncogene alpha transcripts were detected in clusters of keratinocytes of the upper epidermis at the same sites where interleukin-8 mRNA was abundant. In contrast to interleukin-8, growth-related oncogene alpha was also detected in the papillary dermis produced by vessel-associated cells. Sites of interleukin-8 and growth-related oncogene alpha mRNA expression were associated with infiltration of neutrophils. Interestingly, mRNA expression of the highly homologous chemokine ENA-78 was quiescent. In conclusion, our data indicate that growth-related oncogene alpha is an important chemoattractant for neutrophil diapedesis in vivo, whereas further migration of neutrophils and formation of micropustules appears to be influenced by the cooperative action of both growth-related oncogene alpha and interleukin-8.

Differential expression of the small inducible cytokines GRO α and GROβ by synovial fibroblasts in chronic arthritis: Possible role in growth regulation

Synovial pannus represents a hypertrophic and locally invasive connective tissue response to chronic inflammation that accounts in large part for the periarticular destruction of rheumatoid arthritis. Synovial fibroblasts cultured from rheumatoid synovia have been found to display an increased rate of proliferation and the constitutive expression of collagenases, growth factors, and inflammatory cytokines. The existence in rheumatoid synovium of both a pro-inflammatory state and growth dysregulation led us to investigate the expression by synovial fibroblasts of the closely homologous cytokines GROα ( gro MGSA ), GROβ (MIP-2α), and GROγ (MIP-2β). These cytokines are released by a variety of cell types and display overlapping growth regulatory and pro-inflammatory activities. In contrast to expectations, the majority of synovial fibroblasts cell lines derived from osteoarthritic or non-inflammatory synovia showed a relative increase in the constitutive expression of GROα and GROβ when compared to synovial fibroblasts obtained from rheumatoid synovia. Considered together with evidence that GROα is a growth regulator that modulates the expression of metalloproteinase activity, these findings provide evidence for a differential pathway of cytokine activation that may downregulate the proliferative and erosive response to chronic arthritis.

Characterization of GRO α, β and γ expression in human colonic tumours: potential significance of cytokine involvement

The GRO genes, isolated from transformed fibroblasts, belong to a superfamily of genes such as platelet factor 4 and neutrophil activating peptide/IL-8. Three related GRO genes are described which are closely linked on chromosome 4: GROα, GROβ, and GROγ: GROβ and GROγ share 90 and 86% sequence homology with GROα. The GROα gene product shares homology with, and is melanocyte growth stimulatory activity (MGSA). The MGSA/GROα has potent chemotactic, growth regulatory and transformative functions. The function of GROβ and γ is unknown. Expression of GROα is well characterized in vitro: studies in actual human tissues are not reported. We chose to determine the specific expression of GROα, β and γ in both normal and transformed human colonic tissues and to assess the role of exogenous cytokines on their induction. Tissues from ten patients with colonic neoplasia were obtained at the time of colectomy. All specimens underwent Northern analysis for GRO gene expression, comparing normal colonic mucosa with neoplastic mucosa. Differential GROα, β and γ expressions were determined by polymerase chain reaction (PCR). GROα expression was evaluated in the tumour specimens compared with normal, while there was constitutive expression of GROγ in both normal and neoplastic colonic mucosa. Expression of GROβ was minimal in all tissue specimens. In addition, HT29 colon carcinoma cells stimulated with IL-1β and TNFα demonstrated induction of GROα and IL-8. Thus, GROα is differently elevated in in vivo colon carcinoma specimens. GROγ was constitutively expressed in colonic tissues; GROβ was not similarly expressed. Exogenous cytokines such as IL-1β and TNFα may play a role in the specificity of GRO expression, and also stimulate related genes in this superfamily. This overexpression of GROα may contribute to the transformative potential of human colonic carcinoma.

Post-transcriptional regulation of groα, β, γ, and IL-8 mRNAs by IL-1β

Expression of the cytokine gene gro, also known as melanoma growth stimulatory activity, is induced by inflammatory stimuli, including IL-1. To determine whether gro expression is regulated at a post-transcriptional level, the effect of IL-1 on gro mRNA stability was examined. Treatment of fibroblasts with IL-1 beta caused a dose-dependent induction of gro mRNA. When IL-1 was withdrawn, gro mRNA decayed rapidly with a half life of 1 hour. This decay occurred whether or not actinomycin D was added to block new transcription. In contrast, when IL-1 was present in the medium, the level of gro mRNA was stable over 8 hours following addition of actinomycin D. In addition, the stability of a related mRNA, IL-8, was found to be regulated by IL-1. To examine whether Northern results reflected expression of gro alpha, or of the closely related genes, gro beta and gro gamma, RNA samples were analyzed by PCR. All three genes were found to be induced by IL-1 and all mRNAs were stabilized in the presence of IL-1. Northern analysis revealed a minor species of gro mRNA which lacked poly(A). The pattern of expression of this RNA suggested that it was a decay intermediate of one or more of the gro mRNAs. The findings indicate that mRNA stabilization is an important component of IL-1 induced gene expression.