Bent Rolstad

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)

A monoclonal antibody raised against an [Na+K+]coupled l‐glutamate transporter purified from rat brain confirms glial cell localization

A monoclonal antibody (9C4) shows that an [Na+K+]coupled glutamate transporter protein purified from rat brain runs electrophoretically as a wide band and is localized in neuroglial cell bodies and processes, but not in neurons. This confirms the findings with polyclonal antibodies [Neuroscience 51 (1992) 295‐310], and shows that the apparent heterogeneity in relative molecular mass is accounted for by a single antigenic epitope. By testing several synthetic peptides derived from the deduced amino acid sequences of two cloned rat brain glutamate transporters, the antigenic epitope was identified as residing within the peptide TQSVYDDTKNHRESNSNQC (residues 518–536) of one of these [Nature 360 (1992) 464‐467].

MICROBIAL COLONIZATION INFLUENCES COMPOSITION AND T-CELL RECEPTOR VBETA REPERTOIRE OF INTRAEPITHELIAL LYMPHOCYTES IN RAT INTESTINE

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ‐free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T‐cell receptor (TCR) α/β+ IEL in that the proportions of CD4+ and CD8αβ+TCRα/β+ cells were increased, while the double negative (CD4− CD8−) fraction was reduced. Microbial colonization also influenced the TCR Vβ repertoire of CD8+ IEL in that the proportions of Vβ8.2+ and Vβ10+ cells were increased, whereas Vβ8.5+ and Vβ16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same Vβ subsets. Three‐colour flow‐cytometric analysis demonstrated that skewing of the Vβ repertoire was most pronounced in the CD8αα+ subset, although the numerical increase of IEL mainly included the CD8αβ+ subset. In contrast to IEL, the TCR Vβ repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCRα/β+ IEL subpopulations respond dynamically to the microbial gut flora and suggest that their Vβ repertoire can be shaped by luminal microbial antigens.

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.

Selective activation of cAMP-dependent protein kinase type I inhibits rat natural killer cell cytotoxicity.

The present study examines the expression and involvement of cAMP-dependent protein kinase (PKA) isozymes in cAMP-induced inhibition of natural killer (NK) cell-mediated cytotoxicity. Rat interleukin-2-activated NK cells express the PKA α-isoforms RIα, RIIα, and Cα and contain both PKA type I and type II. Prostaglandin E2, forskolin, and cAMP analogs all inhibit NK cell lysis of major histocompatibility complex class I mismatched allogeneic lymphocytes as well as of standard tumor target cells. Specific involvement of PKA in the cAMP-induced inhibition of NK cell cytotoxicity is demonstrated by the ability of a cAMP antagonist, (Rp)-8-Br-adenosine 3′,5′-cyclic monophosphorothioate, to reverse the inhibitory effect of complementary cAMP agonist (Sp)-8-Br-adenosine 3′,5′-cyclic monophosphorothioate. Furthermore, the use of cAMP analog pairs selective for either PKA isozyme (PKA type I or PKA type II), shows a preferential involvement of the PKA type I isozyme, indicating that PKA type I is necessary and sufficient to completely abolish killer activatory signaling leading to NK cell cytotoxicity. Finally, combined treatment with phorbol ester and ionomycin maintains NK cell cytotoxicity and eliminates the cAMP-mediated inhibition, demonstrating that protein kinase C and Ca2+-dependent events stimulate the cytolytic activity of NK cells at a site distal to the site of cAMP/PKA action.

The athymic nude rat: an animal experimental model to reveal novel aspects of innate immune responses?

Summary: Athymic nude rats resemble nude mice in their lack of a normal thymus and functionally mature T cells. They have been useful in the study of mechanisms of tumor growth or graft rejection in immunocompromised hosts since they can accept major histocompatibility complex (MHC) mismatched organ allografts or xenografts for several months and because a number of tumor cell lines of human and rodent origin grow well in these rats. Injection of a few helper T (Th) cells from euthymic littermate rats partly restores the pool of mature T cells as well as full immunocompetence to reject organ allografts and has helped to reveal some of the cell interactions necessary for rejection to occur. In contrast, immunologically naïve athymic nude rats of certain strains, acutely reject allografts consisting of lymphocytes or bone marrow cells, which is due to the presence of alloreactive natural killer cells. These cells can recognize and kill MHC incompatible hematopoietic cells through the recognition of both mismatches within the classical (RT1.A) and non‐classical (RT1.C/E) MHC class I regions with a repertoire of inhibitory and activating killer lectin‐like receptors (KLR) for MHC‐I molecules, encoded by the Ly‐49 portion of the rat natural killer cell gene complex (NKC). Some of these receptors have been identified and molecularly cloned and show similarities with NK receptors identified in the mouse. Other leukocytes in nude rats, such as dendritic cells, may also contribute to specific innate immune responses in the absence of mature T cells. Nude rats develop T‐like cells expressing CD3 and T‐cell receptor (TCR) with increasing age. Though their phenotype in peripheral lymphatic tissues resembles that of normal T cells, consisting mainly of CD4+ or CD8+ cells, they lack alloreactivity in vivo and their TCR repertoire is more of an oligoclonal nature. Their contribution to allograft rejection in T‐cell‐reconstituted rats is therefore questionable, and their role in innate immune response in these rats still enigmatic.

Positive and negative MHC class I recognition by rat NK cells.

Summary: The prompt rejection of transplanted allogeneic lymphocytes by rat NK cells in non‐sensitized recipients (allogeneic lymphocyte cyto‐toxicity or ALC) is determined by MHC genes as well as by genes located in the NK complex. The same genetic control is found when NK alloreactivity is measured by an in vitro assay, and we have employed this assay to delineate the specificity of NK cells for the MHC. The MHC of the rat, RT1, contains class 1 genes situated on either side of the class Il/class III region. The majority of these class 1 genes are located in the RT1.C region and expressed class I products usually behave as non ‐classical (class Ib) molecules. They do not serve as restriction elements for the vast majority of conventional a/p T‐cells, in contrast to those class molecules encoded by one or more loci in the classical (doss la) region, RT1. A. However, NK cells appear to recognize the products of either class 1 region. Immunogenetic studies suggest that NK cells are inhibited by RT1.A molecules, whereas RT1.C region molecules may have a dual role in regulating NK cytolytic activity, i.e. they either inhibit or activate natural killing. Based on THESE premises, a model is proposed in which identification of a target as self or non‐self depends on different receptors for class 1 in single NK cells, interpreting coincident positive and negative signals from the various target class I molecules. The putative role of peptides presented by class I, the biological implications, and the evolution of the NK receptors and ether ligands are discussed.

Pectic polysaccharides from Biophytum petersianum Klotzsch, and their activation of macrophages and dendritic cells.

The Malian medicinal plant Biophytum petersianum Klotzsch (Oxalidaceae) is used as a treatment against various types of illnesses related to the immune system, such as joint pains, inflammations, fever, malaria, and wounds. A pectic polysaccharide obtained from a hot water extract of the aerial parts of B. petersianum has previously been reported to consist of arabinogalactans types I and II (AG-I and AG-II), probably linked to a rhamnogalacturonan backbone. We describe here further structural characteristics of the main polysaccharide fraction (BP1002) and fractions obtained by enzymatic degradations using endo-alpha-d-(1-->4)-polygalacturonase (BP1002-I to IV). The results indicate that in addition to previously reported structures, rhamnogalacturan type II and xylogalacturonan areas appear to be present in the pectic polymer isolated from the plant. Atomic force microscopy confirmed the presence of branched structures, as well as a polydisperse nature. We further tested whether the BP1002 main fraction or the enzymatically degraded products could induce immunomodulating activity through stimulation of subsets of leukocytes. We found that macrophages and dendritic cells were activated by BP1002 fractions, while there was little response of T cells, B cells, and NK cells. The enzymatic treatment of the BP1002 main fraction gave important information on the structure-activity relations. It seems that the presence of rhamnogalacturonan type I is important for the bioactivity, as the bioactivity decreases with the decreased amounts of rhamnose, galactose, and arabinose. The demonstration of bioactivity by the plant extracts might indicate the mechanisms behind the traditional medical use of the plant.

The Novel Inhibitory NKR-P1C Receptor and Ly49s3 Identify Two Complementary, Functionally Distinct NK Cell Subsets in Rats

The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique functional characteristics. NKR-P1C+ NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHC-mismatched lymphoblast target cells. The NKR-P1C+ NK cell subset also appears earlier during development and shows a tissue distribution distinct from its complementary Ly49s3+ subset, which expresses a wide range of Ly49 receptors. These data suggest the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor repertoires.