Autocrine growth effect of IL-8 and GROalpha on a human pancreatic cancer cell line, Capan-1

A human pancreatic cancer cell line, Capan-1, secretes the chemokines interleukin-8 (IL-8) and growth-related oncogene alpha (GROalpha). Capan-1 cells also express the chemokine receptor 2 (CXCR2), which is a Gialpha-protein coupled receptor. Growth of Capan-1 cells was inhibited when anti-IL-8 or anti-GROalpha monoclonal antibody was added into the culture medium. Pertussis toxin, which blocks Gialpha also demonstrated a growth-inhibitory effect on Capan-1 cells. These results indicated that IL-8 and GROalpha act on Capan-1 cells as growth factors in an autocrine manner through CXCR2.     

CXCR1 and CXCR2 silencing modulates CXCL8-dependent endothelial cell proliferation, migration and capillary-like structure formation

CXCR1 and CXCR2 are receptors for angiogenic ELR + CXC chemokines and are differentially expressed on endothelial cells; however, their functional significance in angiogenesis remains unclear. In this study, we determined the functional significance of these receptors in modulating endothelial cell phenotype by knocking-down the expression of CXCR1 and/or CXCR2 in human microvascular endothelial cells (HMEC-1) using short-hairpin RNA (shRNA). Cell proliferation, migration, invasion and capillary-like structure (CLS) formation were analyzed. Our data demonstrate that knock-down of CXCR1 and/or CXCR2 expression inhibited endothelial cell proliferation, survival, migration, invasion and CLS formation. Additionally, we examined the mechanism of CXCL8-dependent CXCR1 and/or CXCR2 mediated phenotypic changes by evaluating ERK phosphorylation and cytoskeletal rearrangement and observed inhibition of ERK phosphorylation and cytoskeletal rearrangement in HMEC-1-shCXCR1, HMEC-1-shCXCR2 and HMEC-1-shCXCR1/2 cells. Together, these data demonstrate that CXCR1 and CXCR2 expression plays a critical role in regulating multiple biological activities in human microvascular endothelial cells.     

VEGF-A,HGF and bFGF are involved in IL-17A-mediated migration and capillary-like vessel formation of vascular endothelial cells

Background: Interleukin (IL)-17A possesses biological activities to promote vascular endothelial cell migration and microvessel development. Objective: To clarify which angiogenic factors are involved in IL-17A-modified angiogenesis-related functions of vascular endothelial cell migration and microtube development or not. Methods: The potential contribution of various angiogenic stimulators to in vitro angiogenic activities of IL-17A was assessed with both modified Boyden Chemotaxicell chamber assay and in vitro angiogenesis assay. Results: The addition of a neutralizing antibody (Ab) for hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF)-A to the upper and lower compartments in a modified Boyden Chemotaxicell chamber significantly attenuated human dermal microvascular endothelial cell (HMVEC) migration elicited by IL-17A. Moreover, IL-17A-induced capillary-like microvessel development in human umbilical vein endothelial cell (HUVEC) and human dermal fibroblast (HDF) co-culture system was significantly impaired by a neutralizing Ab against HGF, bFGF, VEGF-A, cysteine-x-cysteine ligand 8 (CXCL8)/IL-8 or cysteine-x-cysteine (CXC) chemokine receptor (CXCR)-2. Conclusion: Our findings demonstrate the involvement of HGF, bFGF, VEGF-A and/or CXCL8/IL-8, to various degrees, in migration and microvessel development of vascular endothelial cells mediated by IL-17A.     

Upregulation of CXCR1 by proliferating cells in patients with lymphoproliferative disease of granular lymphocytes

The expression and the functional activities of different chemokine receptors (CC motif: CCR1, CCR2, CCR3, CCR5, CCR6; CXC motif: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5) were investigated in 12 patients with lymphoproliferative disease of granular lymphocytes (LDGL). Six patients were characterized by the proliferation of CD3+ve GL and six patients by the expansion of CD3-ve GL. The interleukin 8 (IL-8/CXCL8) receptor CXCR1 was expressed in 12/12 patients, the CXCR4 in 6/12 patients (four CD3+ve and two CD3-ve) and the CXCR3 in 3/12 patients (one CD3+ve and two CD3-ve). CXCR1 was expressed only by proliferating GL. Other CC and CXC receptors were not expressed on proliferating GL (< 2%). In functional assays, purified GL from the patients displayed significant migration in response to specific chemokines, indicating that CXCR1, CXCR3 and CXCR4 were functionally active in these patients. In addition, a significant reduction of IL-8/CXCL8-mediated cell migration was reported in the presence of anti-CXCR1 monoclonal antibody. Our results indicate that expanding cells from patients with LDGL express specific CXCR. These data may help to define functional properties of proliferating GL in patients with LDGL and contribute toward the understanding of the complex clinical features of this disease. In particular, as CXCR1 was expressed in all of the patients studied, we speculate that abnormal expression of this receptor on proliferating GL might play a role in the pathogenesis of neutropenia, which represents a common feature in LDGL patients.     

IL-8 and its CXCR1 and CXCR2 receptors participate in the control of megakaryocytic proliferation, differentiation, and ploidy in myeloid metaplasia with myelofibrosis

Myeloproliferation, myelofibrosis, and neoangiogenesis are the 3 major intrinsic pathophysiologic features of myeloid metaplasia with myelofibrosis (MMM). The myeloproliferation is characterized by an increased number of circulating CD34+ progenitors with the prominent amplification of dystrophic megakaryocytic (MK) cells and myeloid metaplasia in the spleen and liver. The various biologic activities of interleukin 8 (IL-8) in hematopoietic progenitor proliferation and mobilization as well as in neoangiogenesis prompted us to analyze its potential role in MMM. We showed that the level of IL-8 chemokine is significantly increased in the serum of patients and that various hematopoietic cells, including platelets, participate in its production. In vitro inhibition of autocrine IL-8 expressed by CD34+ cells with either a neutralizing or an antisense anti-IL-8 treatment increases the proliferation of MMM CD34(+)-derived cells and stimulates their MK differentiation. Moreover, addition of neutralizing anti-IL-8 receptor (CXC chemokine receptor 1 [CXCR1] or 2 [CXCR2]) antibodies to MMM CD34+ cells cultured under MK liquid culture conditions increases the proliferation and differentiation of MMM CD41+ MK cells and restores their polyploidization. Our results suggest that IL-8 and its receptors participate in the altered MK growth that features MMM and open new therapeutic prospects for this still incurable disease.     

Human CD34(+)CXCR4(-) sorted cells harbor intracellular CXCR4, which can be functionally expressed and provide NOD/SCID repopulation

Homing and repopulation of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice by enriched human CD34(+) stem cells from cord blood, bone marrow, or mobilized peripheral blood are dependent on stromal cell-derived factor 1 (SDF-1)/CXCR4 interactions. Recently, human cord and fetal blood CD34(+)CD38(-)CXCR4(-) and CXCR4(+) cells, sorted with neutralizing anti-CXCR4 monoclonal antibody (mAb), were shown to have similar NOD/SCID repopulation potential. Herein we report that human cord blood CD34(+)CXCR4(+) (R4(+)) and CD34(+)CXCR4(-) (R4(-)) subsets, sorted with neutralizing anti-CXCR4 mAb, engrafted NOD/SCID mice with significantly lower levels of human cells compared with nonsorted and SDF-1-migrated CD34(+) cells. Coinjection of purified cells with 10 microg anti-CXCR4 mAb significantly reduced engraftment of all CD34(+) subsets, and 50 microg completely abrogated engraftment by R4(-) and CD34(+) cells. Importantly, R4(-) cells harbor intracellular CXCR4, which can be rapidly induced to cell surface expression within a few hours. Moreover, 48 hours of cytokine stimulation resulted in up-regulation of both cell surface and intracellular CXCR4, restoring migration capacities toward a gradient of SDF-1 and high-level NOD/SCID repopulation potential. In addition, homing of sorted R4(-) cells into the murine bone marrow and spleen was significantly slower and reduced compared to CD34(+) cells but yet CXCR4 dependent. In conclusion, R4(-) cells express intracellular CXCR4, which can be functionally expressed on the cell membrane to mediate SDF-1-dependent homing and repopulation. Our results suggest dynamic CXCR4 expression on CD34(+) stem and progenitor cells, regulating their motility and repopulation capacities.     

Stromal cells in lymph nodes attract B-lymphoma cells via production of stromal cell-derived factor-1

Stromal cell-derived factor-1 (SDF-1) is a chemokine produced by bone marrow stromal cells which plays an important role in B-lymphopoiesis and the homing of hematopoietic stem cells to the bone marrow. In the present study, we investigated the role of SDF-1 and its receptor, CXCR4, in the chemotactic interaction between non-Hodgkin B-lymphoma cells and lymph node stromal cells. SDF-1 mRNA was abundantly expressed in stromal cells isolated from the lymph nodes of patients with malignant lymphoma. All B-lymphoma cells freshly isolated from these patients and most laboratory B-lymphoma cell lines, including follicular, diffuse large, and Burkitt's lymphoma cells, expressed surface CXCR4 and migrated in the presence of recombinant human SDF-1alpha. Chemotaxis assays revealed that CXCR4-positive (but not CXCR4-negative) B-lymphoma cells migrated towards lymph node stromal cells, and this migration was almost completely inhibited by the addition of anti-CXCR4 monoclonal antibody to the lymphoma cells or of anti-SDF-1 neutralizing antibody to the culture supernatant of the stromal cells. Down-regulation of surface CXCR4 was detected in B-lymphoma cells which migrated towards the stromal cells but not in those which showed no migratory response. In addition, contact between the lymphoma cells and the stromal cells resulted in down-regulation of surface CXCR4 on the lymphoma cells. These data strongly suggest that SDF-1/CXCR4 is the main chemokine system involved in the chemotactic interaction between B-lymphoma cells and lymph node stromal cells.     

The antiviral activity of the CXCR4 antagonist AMD3100 is independent of the cytokine-induced CXCR4/HIV coreceptor expression level

The chemokine receptor CXCR4 is the main coreceptor used by T-tropic X4 HIV-1 strains to infect its target T cells. It has been proven that the CXCR4 expression level in T cells is strongly up-regulated by interleukin (IL)-4, a Th2-type cytokine that is secreted preferentially in HIV-infected patients in a later stage of disease. This results in an enhancement of HIV-1 replication in CD4+ T-lymphocytes. We have now evaluated the potency of the CXCR4 antagonist AMD3100 in phytohemagglutinin (PHA)/IL-2- versus PHA/IL-4-activated T cells in order to determine whether the compound has comparable CXCR4-antagonistic and anti-HIV-1 effects under these different cytokine treatments. We analyzed the CXCR4 expression level and the dose-dependent inhibition of CXCR4 expression by AMD3100, by monitoring the binding of an anti-CXCR4 monoclonal antibody (clone 12G5). We also determined stromal cell-derived factor (SDF)-1-induced intracellular calcium signaling and HIV-1 replication in these cells in the absence and presence of AMD3100. The CXCR4 expression level in PHA/IL-4-stimulated cells was much higher than in PHA/IL-2-stimulated cells. However, the potency of the bicyclam AMD3100 to block anti-CXCR4 mAb binding, SDF-1-induced intracellular calcium signaling, and HIV-1 replication of the X4 NL4.3 strain and three primary isolates remained unchanged. Our data indicate that CXCR4 antagonists such as AMD3100 act independently of the HIV-1 coreceptor expression level. These compounds should therefore be useful in suppressing HIV-1 infection in all stages of the disease.     

A molecular cascade showing nitric oxide-heme oxygenase-1-vascular endothelial growth factor-interleukin-8 sequence in human endothelial cells

Heme oxygenase (HO)-1 has been shown to be an important biological target of nitric oxide (NO). NO can induce HO-1 expression and IL-8 production, particularly, in endothelial cells. Interestingly, HO-1 tends to induce the production of vascular endothelial growth factor (VEGF) that is involved in endothelial IL-8 syntheses. Whether HO-1 expression by NO may provide a link with IL-8 or VEGF synthesis was investigated in human umbilical vein endothelial cells (HUVECs). The NO donor S-nitroso-N-acetyl-penicillamine (SNAP) dose-dependently increased IL-8 and VEGF productions and HO-1 expression in HUVECs. Transfection with either HO-1 small interfering RNA or HO-1 antisense oligodeoxynucleotide abrogated the ability of SNAP to induce HO-1 expression and IL-8 and VEGF productions. Both pharmacological induction and gene transfer of HO-1 directly induced IL-8 and VEGF productions. Anti-VEGF neutralizing antibody blocked SNAP-mediated IL-8 production and VEGF itself induced IL-8 production, whereas anti-IL-8 neutralizing antibody had no effect on VEGF production in SNAP-treated HUVECs. Neither anti-VEGF nor anti-IL-8 antibodies influenced SNAP-induced HO-1 expression. Moreover, neither VEGF nor IL-8 showed an additive effect on SNAP-induced HO-1 expression. HO-1 transfection had no significant effect on productions of other CXC chemokines, such as growth-related oncogen-alpha and epithelial neutrophil activation peptide-78. Taken together, these results provide a molecular cascade showing NO-HO-1-VEGF-IL-8 sequence in human endothelial cells.     

Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells

IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2–induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high levels of IL-2Rβγ, including CD8⁺ T cells and natural killer cells, which resulted in a considerable antitumor response against s.c. and pulmonary B16 melanoma nodules. However, high-dose IL-2 treatment also affected immune cell lineage marker-negative CD31⁺ pulmonary endothelial cells via binding to functional αβγ IL-2Rs, expressed at low to intermediate levels on these cells, thus causing pulmonary edema. Notably, IL-2–mediated pulmonary edema was abrogated by a blocking antibody to IL-2Rα (CD25), genetic disruption of CD25, or the use of IL-2Rβγ–directed IL-2/anti-IL-2 antibody complexes, thereby interfering with IL-2 binding to IL-2Rαβγ⁺ pulmonary endothelial cells. Moreover, IL-2/anti-IL-2 antibody complexes led to vigorous activation of IL-2Rβγ⁺ effector immune cells, which generated a dramatic antitumor response. Thus, IL-2/anti-IL-2 antibody complexes might improve current strategies of IL-2–based tumor immunotherapy.     

Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis

In this study, flow cytometry was used to evaluate interleukin-6 (IL-6) production by bone marrow mononuclear cells from 47 patients with multiple myeloma (MM) in different clinical stages and 15 patients with monoclonal gammopathy of undetermined significance. In patients with MM, autocrine IL-6 production paralleled the clinical disease stage. The largest proportion of syndecan-1(+)/IL-6(+) cells was detected in patients with resistant relapse or primary refractory disease, suggesting that tumor progression involves expansion of myeloma cells producing IL-6. The authors assessed autocrine IL-6 production and in vitro proliferation and apoptosis of myeloma cells in 6 myeloma cell clones (MCCs) and in 2 myeloma cell lines, namely IM-9 and U-266-1970, which showed different sensitivities to the addition of exogenous IL-6. Autocrine IL-6 production was observed in IL-6-independent MCC-2, MCC-3, and MCC-5 cloned from patients with aggressive disease and in the IM-9 cell line. In contrast, IL-6-dependent MCC-1, MCC-4, and MCC-6 were syndecan-1(+) and IL-6(-). Blocking experiments with anti-IL-6 monoclonal antibody from clone AH65, which binds IL-6-IL-6Ralpha complexes, prevented cell proliferation of IL-6(+) MCCs. Flow cytometry evaluations after propidium iodide staining revealed different susceptibilities of MCCs to cell death. IL-6-producing MCCs showed minimal spontaneous and dexamethasone-induced apoptosis, whereas a regular amplitude of apoptosis occurred in the IL-6(-) MCCs. These data provide evidence that autocrine IL-6 reflects a highly malignant phenotype of myeloma cells. In fact, autocrine IL-6 production and deregulated apoptosis may induce expansion of selective IL-6(+) myeloma cells resistant to spontaneous and drug-induced cell death.     

Acquisition of growth autonomy and tumorigenicity by an interleukin 6-dependent human myeloma cell line transfected with interleukin 6 cDNA.

In human multiple myeloma, an autocrine growth mechanism through interleukin 6 (IL-6) has been advocated. However, growth of myeloma cells in vitro is poor except for established cell lines, and IL-6 autocrine growth is quite rare in myeloma cell lines. In the present study, we devised a model of IL-6 autocrine growth in vitro by transfecting IL-6 cDNA into a human myeloma cell line that had a proliferative response to IL-6 but did not produce IL-6. After IL-6 transfection, the cells proliferated in culture media without IL-6, and their growth rate was elevated at higher cell densities. IL-6 was detected by enzyme-linked immunosorbent assay in the culture media of the transfectants. IL-6 mRNA was distinctly expressed in these cells when analyzed by Northern blotting. The growth of the transfectants was definitely inhibited by anti-IL-6 or anti-IL-6 receptor monoclonal antibodies. Furthermore, the transfectants were successfully transplanted to nude mice. These results indicate that the myeloma cells obtained growth autonomy in vitro through IL-6 and tumorigenicity in vivo, after IL-6 transfection.     

Interleukin 1 is an autocrine regulator of human endothelial cell growth.

Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, we demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth and suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells.     

Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo

Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.     

CXCR2 modulates bone marrow vascular repair and haematopoietic recovery post‐transplant

Murine models of bone marrow transplantation show that pre‐conditioning regimens affect the integrity of the bone marrow endothelium and that the repair of this vascular niche is an essential pre‐requisite for successful haematopoietic stem and progenitor cell engraftment. Little is known about the angiogenic pathways that play a role in the repair of the human bone marrow vascular niche. We therefore established an in vitro humanized model, composed of bone marrow stromal and endothelial cells and have identified several pro‐angiogenic factors, VEGFA, ANGPT1, CXCL8 and CXCL16, produced by the stromal component of this niche. We demonstrate for the first time that addition of CXCL8 or inhibition of its receptor, CXCR2, modulates blood vessel formation in our bone marrow endothelial niche model. Compared to wild type, Cxcr2^?/? mice displayed a reduction in bone marrow cellularity and delayed platelet and leucocyte recovery following myeloablation and bone marrow transplantation. The delay in bone marrow recovery correlated with impaired bone marrow vascular repair. Taken together, our data demonstrate that CXCR2 regulates bone marrow blood vessel repair/regeneration and haematopoietic recovery, and clinically may be a therapeutic target for improving bone marrow transplantation.