Search

The in-vitro proliferative reaction of peripheral blood lymphocytes (measured by [³H]thymidine incorporation) to autologous pokeweed mitogen (PWM)-induced lymphoblasts (PWM-lymphoblast-stimulated autologous mixed leucocyte reaction. PWM.AMLR) was used as a measure of immune hyperreactivity for comparison of atopic with non-atopic individuals. Accordingly. 10/24 non-atopics responded in the PWM.AMLR, and (9/19 atopics reacting to inhaled allergens responded. Autologous stimulation was associated with release of mitogenic factors from the PWM-activated stimulating cells (2/15 non-atopics, 9/15 atopics). For non-atopics. stimulation delivered by staphylococcus A (SAC)-activated cells was similar to that delivered by PWM-induced cells, while in atopics. the SAC.AMLR was never more than 50% of the PWM.AMLR. indicating a possible 1 cell component. Separation by panning of the stimulation cells into lymphocyte subsets supported the notion that stimulation involved a cooperation between B and T4+ T cells. It is proposed that a positive PWMAMLR is dependent upon an initial B cell activation followed by the PWM stimulus dependent upon a previous T cell activation, where atopics have more lymphocytes in an activated state than healthy non-atopics. Such a baseline priming may contribute to an innate sensitivity of atopics to environmental allergens. [ABSTRACT FROM AUTHOR]

Agouti protein, a paracrine signaling molecule normally limited to skin, is ectopically expressed in lethal yellow (A(y)) mice, and causes obesity by mimicking agouti-related protein (Agrp), found primarily in the hypothalamus. Mouse attractin (Atrn) is a widely expressed transmembrane protein whose loss of function in mahogany (Atrn(mg-3J)/ Atrn(mg-3J)) mutant mice blocks the pleiotropic effects of A(y). Here we demonstrate in transgenic, biochemical and genetic-interaction experiments that attractin is a low-affinity receptor for agouti protein, but not Agrp, in vitro and in vivo. Additional histopathologic abnormalities in Atrn(mg-3J)/Atrn(mg-3J) mice and cross-species genomic comparisons indicate that Atrn has multiple functions distinct from both a physiologic and an evolutionary perspective.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRzeta/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling.

Attractin is a rapidly upregulated membrane-associated molecule on activated T cells. It is a member of the CUB family of extracellular guidance and development proteins, sharing with them a protease activity similar to that of Dipeptidyl peptidase IV (DPPIV/CD26). Most remarkably, and in sharp contrast to CD26, it is released from the T cell and is presumed to be a major source of a soluble serum-circulating attractin. Genomic sequencing reveals that the soluble form is not a proteolytic product of the membrane form, but is in fact the result of alternative splicing. Recent results prove that the loss of murine membrane attractin results in the mahogany mutation with severe repercussions upon skin pigmentation and control of energy metabolism. In each of these latter instances, there is a strong likelihood that attractin is moderating the interaction of cytokines with their respective receptors. We propose that attractin is performing a similar function in the immune system through capture and proteolytic modification of the N-terminals of several cytokines and chemokines. This regulatory activity allows cells to interact and form immunoregulatory clusters and subsequently aids in downregulating chemokine/cytokine activity once a response has been initiated. These two properties are likely to be affected by the balance of membrane-expressed to soluble attractin.

Agouti protein and agouti-related protein are homologous paracrine signalling molecules that normally regulate hair colour and body weight, respectively, by antagonizing signalling through melanocortin receptors. Expression of Agouti is normally limited to the skin, but rare alleles from which Agouti is expressed ubiquitously, such as lethal yellow, have pleiotropic effects that include a yellow coat, obesity, increased linear growth, and immune defects. The mahogany (mg) mutation suppresses the effects of lethal yellow on pigmentation and body weight, and results of our previous genetic studies place mg downstream of transcription of Agouti but upstream of melanocortin receptors. Here we use positional cloning to identify a candidate gene for mahogany, Mgca. The predicted protein encoded by Mgca is a 1,428-amino-acid, single-transmembrane-domain protein that is expressed in many tissues, including pigment cells and the hypothalamus. The extracellular domain of the Mgca protein is the orthologue of human attractin, a circulating molecule produced by activated T cells that has been implicated in immune-cell interactions. These observations provide new insight into the regulation of energy metabolism and indicate a molecular basis for crosstalk between melanocortin-receptor signalling and immune function.

Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. We have cloned attractin and find that, as in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common gamma cytokine chain. Except for the latter two domains, the overall structure shares high homology with the Caenorhabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.

We have developed a bispecific antibody that recognizes the CD4 and CD29 antigens simultaneously and that was examined for its ability to target CD4+CD29bright T cells. The premise for using bispecific antibody-toxin conjugates was that monovalent binding to one antigen would not result in internalization while binding through both antigens would predispose toward endocytosis and delivery of the toxin to the cell interior. In this study, we show that the bispecific antibody binds monovalently to CD4 and CD29 and bivalently to both antigens. Both monovalent and bivalent binding rendered the target cells sensitive to complement-mediated lysis, demonstrating that this effector modality cannot take advantage of the dual specificity of the antibody. Bivalent binding, however, allowed modulation of more than 60% of the bound antibody off the surface of CD4+CD29bright cells, which we further exploited to deliver a toxin moiety preferentially to CD4+CD29bright cells. Immunoconjugates incorporating blocked ricin (a ricin holotoxin that has its intrinsic galactose-binding sites blocked by chemically linked affinity ligands) preferentially killed CD4+CD29bright cells in vitro by a factor of 25 in comparison with killing of total CD4+ cells in functional assays. Assays on resting PBL demonstrated a similar specificity of the bispecific immunotoxin for CD4+CD29bright cells with a concomitant relative survival of CD4+CD29dim (CD45RA+). We demonstrated that the potency of the immunotoxin is not only a function of its affinity but also of its propensity to internalize, and that this property is influenced by the degree of bivalent binding. These results open up the possibility of engineering bispecific antibody toxin conjugates for use as therapeutic immunotoxins for selective removal of restricted T cell subsets.

CD4 serves as a receptor for MHC class II antigens and as a receptor for the human immunodeficiency virus (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. Although anti-CD4 crosslinking may increase lck activity, the effects of HIV-1 gp120 have been controversial. Activated protein-tyrosine kinases are known to associate with certain intracellular proteins possessing src-homology regions (SH-2 domains) such as phosphatidylinositol 3-kinase (PI 3-kinase). In this paper, we demonstrate that the CD4:p56lck complex associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis of the reaction products demonstrated the presence of phosphatidylinositol 3-phosphate (PI 3-P) and phosphatidylinositol 4-phosphate (PI 4-P), thus indicating that PI 3 and PI 4 kinases associate with CD4-p56lck. The p85 subunit of PI 3-kinase was also detected in anti-CD4 immunoprecipitates by immunoblotting with anti-p85 antiserum. Significantly, p56lck binding to CD4 appears to be necessary for the detection of lipid kinase activity associated with p56lck. Also, anti-HIV gp120 and anti-CD4 crosslinking induced a 10-15-fold increase in levels of both PI 3- and PI 4-kinase activity in anti-CD4 precipitates. Stimulation of CD4-p56lck-linked PI kinases by crosslinked HIV-1 gp120 may play a role in HIV-1-induced immune defects.

We have developed a bispecific antibody that recognizes the CD4 and CD26 antigens simultaneously and that was examined for its ability to target CD4+CD26+T cells. These latter cells constitute the activated component of the CD4+ CD29highCD45RO+ memory T-cell subset that provides help for B-cell Ig synthesis and help for responses against recall antigens. The purified bispecific antibody exhibited an estimated dissociation constant (kd) of 2.4 x 10(-9) mol/L, on comparison with 1.1 x 10(-9) mol/L for anti-CD26, and 1.6 x 10(-10) mol/L for anti-CD4. Surface plasmon resonance was used to show the bifunctional capacity of the antibody. On binding 125I-bispecific antibody to phytohemagglutinin (PHA)-activated T cells, 54.4% of the bound antibody was internalized. This was the result of bispecific binding, because monovalent fragments of anti-CD4 and anti-CD26 were not able to modulate antigen or induce internalization using both a fluorescent assay and an 125I-internalization assay. The ability of the bispecific antibody to be internalized was used to deliver a toxin, blocked ricin, specifically to cells that are CD4+CD26+. The inability of monovalent fragments to be internalized formed the basis for our hypothesis that monovalent binding by the bispecific immunotoxin would not result in internalization. Against resting E+ T cells, the bispecific immunotoxin developed a minimal effect. On preactivating the same cells, using phorbol myristate acetate (PMA)/ionomycin on concanavalin A (ConA) or especially PHA, levels of CD26 were upregulated and the immunotoxin effectively inhibited the ability to provide help for B-cell Ig synthesis while leaving intact the CD4-CD26+ and CD4+CD26- populations; an effect observed both functionally and by phenotype. The bispecific antibody proved to be most effective at inhibiting a heterologous mixed leukocyte reaction. We propose that this reagent may form the basis for the rational design of toxins designed to modulate activated T cells from, or directed against, tissue grafts.

We have previously found that there is a considerable variation in the responses of lymphocytes of normal individuals to autologous pokeweed mitogen (PWM)-induced lymphoblasts (PWM.AMLR) under completely autologous conditions. Having proposed that the reaction might measure an innate sensitivity for B cell hyperreactivity, we measured the PWM.AMLR of a normal group (8/18 positives) and a group of patients with rheumatoid arthritis (RA; 24/25 positives). Responses of the RA group were in general an order of magnitude greater than those of normal controls that generated positive responses, and the responding cells could clearly be shown to be both CD4-bearing and CD8-bearing T cells. T cell-independent B cell mitogen-induced (staphylococcus A) lymphoblasts derived from RA patients were capable of strongly stimulating autologous responder cells, while similarly treated cells of normal controls induced small responses. The staphylococcus A responses were smaller in general than those induced by PWM-induced lymphoblasts. These results support previous results that the degree of activation of both T cells and B cells determines the size of response in the autologous PWM.AMLR and that the PWM.AMLR may be used to determine differing degrees of the in vivo priming of these cells and their relation to clinical lymphocyte hyperreactivity.

Two methods were employed to determine the zygosity of A and B red cells, the rosetting antiglobulin test and the enzyme-linked immunoadsorbent assay (ELISA) technique. By means of the rosetting antiglobulin test, clear differences between AA and AO as well as BB and BO could be shown; however, this method could not define the variation in the amount of the H antigen as a complimentary means of differentiating AA from AO and BB from BO. The ELISA test could quantitatively estimate the level of expression of H antigen, and using anti-A and anti-B reagents in a simultaneous assay, a specific pattern could be generated for each phenotype. Both rosette and ELISA approaches offer easy and inexpensive means of determining zygosity within the ABO system for use in paternity exclusion.

This report questions the nature of stimulation in the lymphoblast-induced autologous mixed leukocyte reaction (AMLR). Using immobilized phytohemagglutinin (PHA) and pokeweed mitogen (PWM), we show that the AMLR generated with PHA lymphoblasts (PHA X AMLR) was not significantly different from the AMLR generated with untreated stimulators. The PWM lymphoblasts of 15 out of 33 apparently normal blood donors generated an AMLR (PWM X AMLR) greater than their respective normal AMLR. The positive PWM X AMLR was not related to the expression of HLA-DR or surface IgM, since expression of both was increased by both PHA and PWM, yet only PWM blasts stimulated in the AMLR. Fixation of PWM-stimulated cells prior to the AMLR completely abolished stimulatory capacity, indicating further against new or increased antigen expression. Inactivation by uv irradiation of surface HLA-D on the stimulators had no effect upon the PWM X AMLR, while intact protein synthesis was required in order to stimulate. The ability of cells to stimulate was associated with the release of soluble helper factors capable of stimulating autologous cells independently. These factors were neither contaminating PWM nor secreted IL-1 or IL-2, although IL-1-like activity was released by all cells regardless of their ability to stimulate. The individual variation in the PWM X AMLR response and secretion of helper factors is discussed in relation to B-cell hyperproliferation and altered immunoglobulin production in autoimmune manifestations.

An imbalance of osteoblastic bone formation and osteoclastic bone resorption is thought to be responsible for the osteoporotic condition. The pathological events leading to this disorder are, in most instances, uncertain. A defect in the ability of osteoporotic patients to respond to foreign histocompatibility antigens in a mixed leukocyte reaction is reported here. The results further show that this defect is due both to a poorly responding lymphocyte population as well as to a suppressor factor in osteoporotic sera. In addition, there is a significant increase in the relative and absolute numbers of T cells in the patients' peripheral blood, while serum IgG, IgA, and IgM remain within the normal range. These findings are discussed in the light of a common immunopathological pathway regulating osteoclastic activity and leading to the osteoporotic condition.

A competitive repopulation assay utilizing chromosome markers was used to assay the reconstituting potential of hematopoietic populations. The test populations consisted of tibial murine marrow locally irradiated with doses ranging from 1.5 Gy to 8.5 Gy and of marrow generated from either murine splenic or marrow stem cells. The purpose of this assay was to assess the innate proliferative potential and microenvironmental influences on the ability to repopulate. Regardless of origin, spleen repopulating ability consistently agreed with spleen colony-forming unit (CFU-s) content. Doses of radiation from 5 Gy to 8.5 Gy diminished, by a factor of 2, the ability to repopulate marrow despite maintenance of CFU-s levels. Marrow generated from splenic stem cells had one-fifth the repopulating ability of marrow derived from marrow stem cells, even though CFU-s levels were equivalent. The results imply that the splenic environment can only maintain stem cells at the level of the CFU-s, even if the stem cells were originally of higher quality, and that their original potential cannot be regained in a marrow environment. Nevertheless, the marrow can maintain more primitive stem cells, but this reserve is drained to support CFU-s levels.

Accelerated bone loss often accompanies the menopause, and may also affect a significant proportion of ageing women. This osteoporosis can also be an accompaniment of several other pathological manifestations. It is difficult to imagine that each process alters bone resorption and renewal by a separate mechanism; in all probability a common mechanism may operate in some of the instances where osteoporosis is observed. It is proposed that chronic alterations in the levels of circulating physiologically-active cortisol could account for some of the osteoporoses. These alterations need not necessarily affect total plasma cortisol values, but rather affect the relationship of cortisol to its plasma carrier protein, transcortin, since changes in this relationship are a common occurrence in many of the conditions associated with osteoporosis.