Azzam A. Maghazachi

Suppressive Effect of 1α,25-Dihydroxyvitamin D3 on Type I IFN-Mediated Monocyte Differentiation into Dendritic Cells: Impairment of Functional Activities and Chemotaxis

Dendritic cells (DCs) generated by a single-step exposure of human monocytes to type I IFN and GM-CSF (IFN-DCs) are endowed with potent immunostimulatory activities and a distinctive migratory response to specific chemokines. In this study, we evaluated the effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active metabolite of vitamin D3, on the DC differentiation/activation induced by type I IFN. We found that 1,25(OH)2D3 prevented the generation of IFN-DCs when added to freshly isolated monocytes, and was capable of redirecting already differentiated IFN-DCs toward a more immature stage, as revealed by their immunophenotype, reduced allostimulatory activity, and impaired LPS-induced production of Th1-polarizing cytokines. Control and 1,25(OH)2D3-treated IFN-DCs exhibited a similar expression of vitamin D receptor, as well as comparable cell death rates. Furthermore, the chemotactic response of IFN-DCs to CCL4 and CCL19 was markedly reduced or completely abrogated by 1,25(OH)2D3. Despite these changes in the IFN-DC migratory behavior, the expression of CCR5 and CCR7 and the calcium fluxes triggered by CCL4 and CCL19 were not affected. These findings indicate that, in this innovative single-step DC generation model from monocytes, the suppressive effect of 1,25(OH)2D3 is associated with a potent impairment of DC migration in response to inflammatory and lymph node-homing chemokines, thus unraveling a novel mechanism involved in 1,25(OH)2D3-mediated immunomodulation.

Human NK Cells Express CC Chemokine Receptors 4 and 8 and Respond to Thymus and Activation-Regulated Chemokine, Macrophage-Derived Chemokine, and I-309

NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.

Role of Chemokines in the Biology of Natural Killer Cells

Natural killer (NK) cells represent a major subpopulation of lymphocytes. These cells have effector functions as they recognize and kill transformed cells as well as microbially infected cells. In addition, alloreactive NK cells have been successfully used to treat patients with acute myeloid leukemia and other hematological malignancies. NK cells are also endowed with immunoregulatory functions since they secrete cytokines such as IFN-γ, which favor the development of T helper 1 (Th1) cells, and chemokines such as CCL3/MIP-1α and CCL4/MIP-1β, which recruit various inflammatory cells into sites of inflammation. In human blood, NK cells are divided into CD56(bright) CD16(dim) and CD56(dim) CD16(bright) subsets. These subsets have different phenotypic expression and may have different functions; the former subset is more immunoregulatory and the latter is more cytolytic. The CD56(bright)CD16(dim) NK cells home into tissues such as the peripheral lymph nodes (LNs) under physiological conditions because they express the LN homing receptor CCR7 and they respond to CCL19/MIP-3β and CCL21/SLC chemokines. They also distribute into adenoid tissues or decidual uterus following the CXCR3/CXCL10 or CXCR4/CXCL12 axis. On the other hand, both NK cell subsets migrate into inflammatory sites, with more CD56(dim)CD16(bright) NK cells distributing into inflamed liver and lungs. CCR5/CCL5 axis plays an important role in the accumulation of NK cells in virally infected sites as well as during parasitic infections. CD56(bright)CD16(dim) cells also migrate into autoimmune sites such as inflamed synovial fluids in patients having rheumatoid arthritis facilitated by the CCR5/CCL3/CCL4/CCL5 axis, whereas they distribute into inflamed brains aided by the CX₃CR1/CX₃CL1 axis. On the other hand, CD56(dim)CD16(bright) NK cells accumulate in the liver of patients with primary biliary disease aided by the CXCR1/CXCL8 axis. However, the types of chemokines that contribute to their accumulation in target organs during graft vs. host (GvH) disease are not known. Further, chemokines activate NK cells to become highly cytolytic cells known as CC chemokine-activated killer (CHAK) cells that kill tumor cells. In summary, chemokines whether secreted in an autocrine or paracrine fashion regulate various biological functions of NK cells. Depending on the tissue and the chemokine secreted, NK cells may ameliorate the disease such as their roles in combating tumors or virally infected cells, and their therapeutic potentials in treating leukemias and other hematological malignancies, as well as reducing the incidence of GvH disease. In contrast, they may exacerbate the disease by damaging the affected tissues through direct cytotoxicity or by the release of multiple inflammatory cytokines and chemokines. Examples are their deleterious roles in autoimmune diseases such as rheumatoid arthritis and primary biliary cirrhosis.

Interferon-inducible protein-10 and lymphotactin induce the chemotaxis and mobilization of intracellular calcium in natural killer cells through pertussis toxin-sensitive and -insensitive heterotrimeric G-proteins.

We show here that interferon‐inducible protein‐10 (IP‐10), an ELR lacking CXC chemokine, and the C chemokine lymphotactin (Ltn) induce the chemotaxis and calcium mobilization in IL‐2‐activated NK (IANK) and CC chemokine‐activated NK (CHAK) cells. Cross‐desensitization experiments show that IP‐10 or Ltn use receptors not shared by other C, CC, or CXC chemokines. The chemotaxis induced by either IP‐10 or Ltn for both cell types is inhibited upon pretreatment of these cells with pertussis toxin (PT). Also, Ltn‐induced [Ca2+]i in IANK but not in CHAK cells is inhibited upon pretreatment with PT, whereas IP‐10‐induced [Ca2+]i in IANK and CHAK cells is inhibited upon pretreatment with this toxin. These results suggest important roles for PT‐sensitive and ‐insensitive G‐proteins in IP‐10‐induced and Ltn‐induced chemotaxis and calcium fluxes in activated NK cells. This was further implicated after streptolysin O permeabilization of CHAK and IANK cells and after introduction of inhibitory antibodies to the PT‐sensitive Gi and Go or the PT‐insensitive Gq. Our results suggest that IP‐10 and Ltn receptors are coupled to Gi, Go, and Gq in IANK cells and to Gi and Gq in CHAK cells, with a possible low coupling of IP‐10, but not of Ltn, receptors to Go in these cells. Together, these results show that IP‐10 and Ltn‐dependent chemotaxis and calcium mobilization may differentiate at the level of receptor coupling to the heterotrimeric G‐proteins.—Maghazachi, A. A., Skålhegg, B. S., Rolstad, B., Al‐Aoukaty, A. Interferon‐inducible protein‐10 and lymphotactin induce the chemotaxis and mobilization of intracellular calcium in natural killer cells through pertussis toxin‐sensitive and ‐insensitive heterotrimeric G‐proteins. FASEB J. 765–774 (1997)

Functional Expression of H4 Histamine Receptor in Human Natural Killer Cells, Monocytes, and Dendritic Cells

We describe here the protein expression of H4 histamine receptor in cells of the innate immune system, which include NK cells, monocytes, and dendritic cells (DCs). Anti-H4R specifically stained permeabilized NK cells, THP-1 clone 15 monocytes, and DCs. This binding was inhibited by incubating anti-H4R Ab with its corresponding peptide. Histamine induced NK cells, THP-1 clone 15 cells, and DCs chemotaxis with high affinity. The ED50 chemotactic effect was 5 nM, 6.8 nM, and 2.7 nM for NK cells, THP-1 clone 15 cells, and DCs, respectively. Thioperamide, an H3R/H4R antagonist, inhibited histamine-induced chemotaxis in all these cells. However, histamine failed to induce the mobilization of [Ca2+]i in NK cells and THP-1 clone 15 cells, but it induced calcium fluxes in DCs. Using a new method of detecting NK cell-mediated cytolysis, it was observed that NK cells efficiently lysed K562 target cells and that histamine did not affect this NK cell activity. In summary, this is the first demonstration of the protein expression of H4 receptor in NK cells. Also, the results of the chemotactic effects of histamine on NK cells and THP-1 cells are novel. These results may shed some light on the colocalization of cells of innate immune arm at sites of inflammation. They are also important for developing drugs that target H4R for the treatment of various disorders, such as autoimmune and immunodeficient diseases.

MIP-3alpha, MIP-3beta and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells.

We demonstrate here that the CC chemokines macrophage inflammatory protein‐3α (MIP‐3α), macrophage inflammatory protein‐3β (MIP‐3β) and the CX3C chemokine fractalkine induce the chemotaxis of interleukin‐2 (IL‐2)‐activated natural killer (IANK) cells. In addition, these chemokines enhance the binding of [γ‐35S]guanine triphosphate ([γ‐35S]GTP) to IANK cell membranes, suggesting that receptors for these chemokines are G protein‐coupled. Our results show that MIP‐3α receptors are coupled to Go, Gq and Gz, MIP‐3β receptors are coupled to Gi, Gq and Gs, whereas fractalkine receptors are coupled to Gi, and Gz. All three chemokines induced a robust calcium response flux in IANK cells. Cross‐desensitization experiments show that MIP‐3α, MIP‐3β or fractalkine use receptors not shared by each other or by the CC chemokine regulated on activation, normal, T‐cell expressed, and secreted (RANTES), the CXC chemokines stromal‐derived factor‐1α (SDF‐1α) and interferon‐inducible protein‐10 (IP‐10), or the C chemokine lymphotactin.

Sphingosine 1 phosphate induces the chemotaxis of human natural killer cells. Role for heterotrimeric G proteins and phosphoinositide 3 kinases

We have examined the effect of sphingolipids on the chemotaxis of human natural killer (NK) cells. Messenger RNA for Edg‐1, Edg‐6 and Edg‐8 but not Edg‐3, are expressed in these cells. Sphingosine 1 phosphate (SPP), dihydro SPP (DHSPP) or the CC chemokine RANTES (CCL5), but not sphingosine induces the chemotaxis of these cells. Pertussis toxin inhibits the chemotaxis induced by these ligands. Permeabilization of NK cells with streptolysin O (SLO) and introduction of blocking antibodies to the heterotrimeric G proteins, showed that Gαi2, Gαs, Gαq/11 or Gα13 mediate the chemotaxis of SPP, whereas Gαi2, Gαo or Gαq/11 mediate the chemotaxis of DHSPP. Gαi2, Gαo, Gαs, Gαq/11, Gαz or Gα12 mediates RANTES‐induced NK cell chemotaxis. Further analysis showed that phosphoinositide 3 kinase (PI3K) inhibitors wortmannin and LY294002 inhibit NK cell chemotaxis induced by SPP, DHSPP or RANTES. Blocking antibody to PI3Kγ inhibits the chemotaxis induced by the three ligands, whereas anti‐PI3Kβ was without effect. In contrast, SPP and DHSPP recruit PI3Kβ isozyme into NK cell membranes, suggesting that although this isoform is not involved in chemotaxis, it is activated by these phospholipids.

G protein-coupled receptors in natural killer cells.

Natural killer (NK) cells are capable of killing tumor as well as virally infected cells. How these cells migrate toward the infected sites in the body is not completely understood. Chemokine receptors that belong to the heptahelical family of receptors and characteristically bind heterotrimeric G proteins are present in most NK cells. Recent results showed that resting NK cells highly express constitutive chemokine receptors (CCR4, CCR7, CXCR4, and CX3CR1) with low expression of a limited repertoire of inflammatory chemokine receptors (CCR1 and CXCR3). However, only a subset of these cells expressing the CD56dim and adhesion moleculehigh phenotype is capable of in vivo binding to vascular endothelium. Under pathological conditions where inflammatory cytokines are present, these cells are induced to express inflammatory chemokine receptors. Resting as well as activated NK cells also express receptors for another member of the heptahelical family of receptors that bind phosphorylated or glycosylated lysolipids. These include sphingosine 1‐phosphate (S1P)1, S1P4, and S1P5, the receptors for S1P; lysophosphatidic acid (LPA)1, LPA2, and LPA3, the receptors for LPA; and T cell death‐associated gene 8, the receptor for psychosine. Similar to chemokines, S1P, LPA, and psychosine induce the chemotaxis of NK cells through heterotrimeric G proteins. However, in contrast to chemokines, which enhance the cytotoxicity of NK cells, lysolipids inhibit this function. We hope that gaining knowledge regarding the distribution of activated NK cells toward the sites of tumor growth or virally infected sites will give an advantage in designing strategies using these cells as tools for the prevention and treatment of immunodeficiencies.

Chemokines activate natural killer cells through heterotrimeric G-proteins: implications for the treatment of AIDS and cancer

Natural killer (NK) cells are anti‐tumor and anti‐viral effector cells. These cells show increased cytolytic activity upon stimulation with interleukin 2 or chemokines. In addition, members of the C, CC, CXC, or CX3C chemokines induce the in vitro chemotaxis of NK cells and contribute to their in vivo tissue accumulation. Chemokines induce various intracellular signaling pathways in NK cells by activating members of the heterotrimeric G‐proteins. Understanding these pathways should provide an insight into NK cell activation, in vivo distribution, and tissue localization. Based on evidence showing the high lytic activity of these effector cells against transformed or virally infected cells, it is suggested that NK cells can be used to maximize the immunotherapeutic protocols for AIDS and cancer patients.—Maghazachi, A. A., Al‐Aoukaty, A. Chemokines activate natural killer cells through heterotrimeric G‐proteins: implications for the treatment of AIDS and cancer. FASEB J. 12, 913–924 (1998)

IL‐6 and IL‐8 release is mediated via multiple signaling pathways after stimulating dendritic cells with lysophospholipids

Lysophosphatidic acid (LPA) and sphingosine 1‐phosphate (S1P) are bioactive lipid mediators, which are known to play major roles in allergic reactions as well as in tumor pathogenesis. Here, the biological activities and signal pathways of these lysophospholipids (LPLs) in dendritic cells (DCs) were characterized further. Flow cytometric and immunoblot analyses indicate that immature as well as mature DCs express the LPL receptors S1P1, S1P3, S1P5, and LPA2, but not S1P2, S1P4, LPA1, or LPA3. Moreover, enzyme‐linked immunosorbent assay experiments demonstrate that simultaneous addition of these LPLs to immature DCs in the presence of lipopolysaccharide enhanced the secretion of the inflammatory cytokines interleukin (IL)‐6 and IL‐8 in maturing DCs. In contrast, no modification of IL‐6 or IL‐8 release was observed after exposure of mature DCs to LPLs alone. In addition, studies with pertussis toxin and mitogen‐activated protein kinase (MAPK) kinase inhibitor PD98059 suggested that Gi proteins and MAPK pathway are involved in these LPL‐induced cell responses. Corroborating these findings, we observed that LPLs induce the phosphorylation of extracellular signal‐regulated kinase 1/2 in immature DCs but not in mature DCs. Further analyses show that inhibitors of phosholipase D, Rho, and protein kinase C also inhibited the LPL‐induced release of IL‐6 and IL‐8. Therefore, our findings suggest that lipopolysaccharide in DCs uncouples LPL receptors from the signal‐transducing machinery during maturation and that exposure of LPLs at early time‐points to maturing DCs modifies the proinflammatory capacity of mature DCs.