J. Laning

Production and function of monocyte chemoattractant protein-1 and other β-chemokines in murine glial cells

Monocyte chemoattractant protein-1 (MCP-1), formerly termed JE, is a member of the beta-chemokine (C-C chemokine) family and has been shown to be produced by a variety of cell types. Recently, mRNA of JE/MCP-1 was detected in astrocytes during the acute phase of experimental allergic encephalomyelitis (EAE). In addition, supernatants collected from human cultured astrocytes have recently been found to be chemotactic for monocytes. However, chemokine production and function in glial cells has not been fully examined. Using a sandwich ELISA assay, we have now quantitated MCP-1 levels and assessed MCP-1 function on murine glial cells. Lipopolysaccharide (LPS), interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha induced MCP-1 secretion by astrocytes, but not microglia. In addition, pretreatment with interferon (IFN)-gamma significantly augmented MCP-1 production by either LPS or the above cytokines. In contrast, LPS preferentially induced production of another beta-chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha) from microglial cells. MCP-1 induced chemotaxis of microglial cells and macrophages. Similarly, another beta-chemokine, TCA3, which is produced by encephalitogenic T lymphocytes, also induced chemotaxis of microglia and macrophages. These findings suggested that astrocytes and microglial cells differentially produce chemokines in the central nervous system, and that both astrocytes and T cells may facilitate recruitment and activation of microglial cells via production of beta-chemokines.

In vitro analysis of CD40-CD154 and CD28-CD80/86 interactions in the primary T-cell response to allogeneic nonprofessional antigen presenting cells

Background. Recently, several ligand interactions have been examined indetail as potential mediators of costimulatory signaling. The CD154/CD40 andCD28/B7 interactions have been highlighted as being among the more-significantcontributors to proper activation of unprimed T lymphocytes. Humankeratinocytes (HK) and human dermal fibroblasts (HF) are capable of expressingClass II HLA and CD40 antigens after interferon-&ggr; exposure, yet neitherexpress significant levels of B7. HK and HF have been characterized as“nonprofessional” antigen presenting cells (APC) and their poorAPC function has been partially attributed to deficient costimulatoryactivity. Methods. In this study, we examined whether substituting forcostimulatory signaling events through the addition of cross-linked monoclonalantibodies against the T-cell ligand/s (CD28 and/or CD154) could restoreallostimulation. Mixed lymphocyte reactions were performed combining enrichedhuman peripheral blood T cells and allogeneic HK or HF with or withoutstimulatory anti-CD28 and/or anti-CD154antibodies. Results. The results show that the addition of anti-CD28 alonepermitted HF but not HK to present alloantigen effectively. In contrast,addition of both anti-CD154 and anti-CD28 was required to generate even amoderate proliferative response to allogeneic HK. Further, adding amonomorphic anti-HLA-DR antibody substantially inhibited these responses.Additional experiments suggest that signaling through CD40/CD154 directs HK toproduce TGF-&bgr;, which would adversely affect T-cellactivation. Conclusions. The data presented highlight significant differences insignaling capacities for HK versus HF and provide evidence for a partialmechanism by which allogeneic human skin equivalents might be immunologicallynull uponengraftment.

The role of chemokines in transplant immunology

Abstract In this review, we discuss evidence that chemokines play an essential role in regulating and co-ordinating the infiltration of leukocytes into allografts. Chemokines and chemokine receptor-expressing leukocytes are present in allografts, and dysregulation of one or many of these interactions has been shown to inhibit recruitment, as well as the rejection process. Most compelling are observations in experimental models that the chemokine receptors CCR1, CCR5, and CXCR3 are of functional importance. In addition, human transplant recipients with genetic polymorphisms encoding nonfunctional chemokine receptors, especially CCR5, have a better long-term graft survival as compared with that in individuals with functional receptors. Analysis of these polymorphisms and the patterns of expression of intragraft chemokines and their receptors may serve as sensitive markers to identify patients at risk of rejection or other forms of clinical graft dysfunction.

THE RELATIONSHIP OF Mlsx TO Mlsc

Among T cell clones with specificity for cow insulin and autologous class II MHC products, a significant number displayed interesting patterns of alloreactivity to non‐MHC antigens. Four clones are described in this report. One is a typical Mlsa‐reactive clone, while the other three proliferate to a variety of allogeneic spleen cells with reportedly different Mls phenotypes. These include PL/J stimulator cells, designated Mlsx all strains reported to be Mlsc, and several strains previously typed as Mlsa. Little is known about Mlsx except that it does not appear to be cross‐reactive with Mlsa. In this report, therefore, we attempt to investigate the reasons why these clones seem to be stimulated by a variety of different Mls phenotypes. Our conclusions are, first, that some of the strains previously typed as Mlsa may actually express a second Mls product, either c or x, in a manner analogous to the CBA/J strain (which expressess both Mlsa and Mlsc), and second, that Mlsc and Mlsx are cross‐reactive. In preliminary experiments, we investigate the genetic relationship between Mlsc and Mlsx by analysis of backcrosses, and the extent of cross‐reactive recognition of Mlsc and Mlsx by raising T cell clones which recognize one but not the other. Our preliminary conclusion is that Mlsc and Mlsx are cross‐reactive, but represent distinct gene products.

Heterogeneity of T cell clones specific for an encephalitogenic determinant of myelin proteolipid protein (PLP)

ENCEPHALITOGENIC DETERMINANT OF MYELIN PROTEOLIPID PROTEIN (PLP) Vi jay K. Kuchroo, Raymond A. Sobel, Joseph C. Laning, E d w a r d Greenfield, Shyre-Te Ju , Mar t in E. Dorf, and Mar jo r l e B. Lees Harvard Medical School, Boston, MA, Massachusetts General Hospital, Boston, MA, the E.K. Shriver Center, Waltham, MA and Boston University School of Medicine, Boston, MA 02118. Protoolipid protein (PLP) is the major protein of central nervous system myelin. SJL (H-2 s) mice immunized with a synthetic peptide corresponding to PIP residues 139-151 develop acute EAE. In this study, 7 IA s restricted, CD4 + TcR o~15 bearing T cell clones were derived from SJL/J mice following immunization with this synthetic peptide. The clones responded strongly, in vitro proliferative assays, to the whole PIP molecule and to PLP peptide 139-151, but not to irrelevant antigens. They also responded to truncated and overlapping forms of the peptide. However, at least 5 distinct reactivity patterns were found with the truncated peptides, suggesting that multiple epitopes may exist within the encephalitogenic determinant. Using a panel of anti-TcR VI~ monoclnnal antibodies and by molecular cloning, TcR VI5 usage of the clones was determined. Four clones were found to use 4 different TcR ViPs (VI52, Vp6, V~10 and VI317a), whereas the V~s on three other clones have not been identified. Among the 7 clones tested for induction of EAE following adoptive transfer into syngeneic mice, 5 clones induced disease of varying severity in naive mice or in mice pre-treated with irradiation and pertussis. The encephalitogenic clones were cytolytic against peptide pulsed, IA s bearing targets whereas non-encephalitogenic T cells showed no cytotoxicity. Thus the clones showed diversity in their pattern of epitope recognition, TcR VI~ usage, encephalitogenic potency and cytolytic activity demonstrating that the T cell response to encephalitogenic PLP-determinants is heterogenons. (Supported by NIH NS 26773, NS 16945, CA 39790, MD 04147, and National MS Society RG 2320A-I)

SYNERGY BETWEEN STIMULATOR CELLS IN THE INDUCTION OF THE ANTI‐Mlsa RESPONSE

We have identified two types of clones responsive to Mls determinants. One type responded vigorously to purified B cells from mice bearing Mlsa‐stimulatory determinants. The other type, including clone Lyl‐N5, responded vigorously to unfractionated spleen cells, but failed to respond to B cells alone or to spleenadherent cells (SAC) alone from the Mlsa‐bearing mice. Synergy between two stimulator cell types, B cells and SAC, was required to induce the Mls response of clone Lyl‐N5. The failure of clone Lyl‐N5 to respond to Mlsa‐bearing B cells was reversed by the addition of SAC taken from mice bearing the Mlsb allele or the non‐stimulatory Mlsb allele. B cells were required to provide the Mlsa determinant.