Functional differences between monocyte chemotactic protein-1 receptor A and monocyte chemotactic protein-1 receptor B expressed in a Jurkat T cell.

The monocyte chemotactic protein-1 (MCP-1) receptor (MCP-1R) is expressed on monocytes, a subpopulation of memory T lymphocytes, and basophils. Two alternatively spliced forms of MCP-1R, CCR2A and CCR2B, exist and differ only in their carboxyl-terminal tails. To determine whether CCR2A and CCR2B receptors function similarly, Jurkat T cells were stably transfected with plasmids encoding the human CCR2A or CCR2B gene. Nanomolar concentrations of MCP-1 induced chemotaxis in the CCR2B transfectants that express high, intermediate, and low levels of MCP-1R. Peak chemotactic activity was shifted to the right as receptor number decreased. Five-fold more MCP-1 was required to initiate chemotaxis of the CCR2A low transfectant, but the peak of chemotaxis was similar for the CCR2A and CCR2B transfectants expressing similar numbers of receptors. MCP-1-induced chemotaxis was sensitive to pertussis toxin, implying that both CCR2A and CCR2B are G(i)alpha protein coupled. MCP-1 induced a transient Ca(2+) flux in the CCR2B transfectant that was partially sensitive to pertussis toxin. In contrast, MCP-1 did not induce Ca(2+) flux in the CCR2A transfectant. Since MCP-1 can stimulate chemotaxis of the CCR2A transfectant without inducing Ca(2+) mobilization, Ca(2+) flux may not be required for MCP-1-induced chemotaxis in the Jurkat transfectants. These results indicate that functional differences exist between the CCR2A and CCR2B transfectants that can be attributed solely to differences in the carboxyl-terminal tail.

Vitamin E supplementation of cattle and shelf-life of beef for the Japanese market.

Feeder steers (n = 84) were stratified into four weight groups to provide slaughter groups so that product that had been in vacuum packages at 0 to 2 degrees C for 40, 60, 80, or 100 d postmortem could be simultaneously evaluated. Each of the four groups was randomly divided into three subgroups so that vitamin E could be supplemented in the diet at rates of 0, 1,000, or 2,000 (E0, E1000, and E2000, respectively) IU.steer-1.d-1 for 100 d. After slaughtering, chilling, and fabricating, one ribeye-roll and one strip loin from each carcass was transported to the university laboratory for analyses, whereas the paired subprimals were transported to Japan. Based on metmyoglobin formation and lipid oxidation, strip loin steaks deteriorated at a faster rate during retail-display than did ribeye steaks. Steaks from subprimals that were stored for 100 d had inferior (P < .05) retail-display characteristics and a shorter (P < .05) caselife than steaks from the other storage periods. alpha-Tocopherol levels in longissimus muscle were lower (P < .05) for E0 than for E1000 and E2000 (3.51, 5.54, and 6.10 micrograms/g of tissue, respectively). Supplementing cattle with vitamin E resulted in steaks that exhibited superior lean color, less surface discoloration, more desirable overall appearance, and less lipid oxidation during retail display than control steaks; minimal differences were observed between E1000 and E2000 steaks. Steaks from cattle supplemented with vitamin E were preferred over control steaks by 91% of Japanese survey participants (n = 10,941), and 58% of all participants identified muscle color as the most important factor in selecting beef products.

Regulation of anxiety by GABAA receptors in the rat amygdala.

Blockade of GABAergic inhibition in the region of the anterior basolateral amygdala (BLA) of rats elicits physiologic changes associated with a defense reaction. The present study was undertaken to determine whether GABA receptors in the BLA might be involved in regulating experimental anxiety using the social interaction (SI) and conflict test. Guide cannulae were stereotaxically implanted bilaterally in the BLA of rats for intracerebral microinjections. In the BLA, injection of the GABAA receptor antagonists bicuculline methiodide (BMI) and picrotoxin (PIC) produced anxiogenic-like effects in the SI paradigm, as did BMI injection using the conflict paradigm. Injection of the GABAA agonist muscimol (MUS) into the central nucleus of the amygdala (Ce) produced anxiolytic-like effects in the SI test. Microinjection of MUS, baclofen (GABAB agonist), 2OH-saclofen (GABAB antagonist) or strychnine (glycine antagonist) into the BLA or BMI into the Ce elicited no change in experimental anxiety as measured by the SI test. These results suggest that endogenous GABA acts tonically at GABAA receptors in the BLA to inhibit anxiety responses.

Priming of experimental anxiety by repeated subthreshold GABA blockade in the rat amygdala.

Blockade of GABAA receptor function in the area of the anterior basolateral amygdala of rats elicits physiological (increases in heart rate and blood pressure) and behavioral changes similar to symptoms of human anxiety states. Repeated subthreshold blockade of GABAA receptors in this region appears to result in a long-term 'priming' of these anxiety-like responses. The present study was conducted to characterize the 'priming' of the heart rate and blood pressure responses and to test if these 'primed' animals would show increases in anxiety responses. Male Wistar rats with arterial catheters placed for physiological measurements were implanted with chronic microinjection cannulae in the anterior basolateral amygdaloid nucleus (BLA) under pentobarbital anesthesia. Repeated daily injections of a subthreshold dose of bicuculline methiodide (GABAA receptor antagonist; BMI) into the BLA elicited 'priming' of physiological responses after 3-5 injections and this response was maintained for at least 6 weeks. The primed animals also showed increased anxiogenic responses to GABAA blockade in the BLA. The 'priming' of anxiety responses were clearly elicited before kindling of seizures as measured by EEG. These results suggest that this 'priming' phenomenon may be similar to kindling and long-term potentiation. This could be one potential mechanism for developing pathological emotional responses, such as chronic, high levels of anxiety.

Anxiolytic effects of chlordiazepoxide blocked by injection of GABAA and benzodiazepine receptor antagonists in the region of the anterior basolateral amygdala of rats.

The amygdala is a structure that is often implicated in the regulation of anxiety responses. Many studies have shown that injection of benzodiazepines into the amygdala produces an anxiolytic effect. The present study was undertaken to determine whether the anxiolytic effect of administration of systemic benzodiazepine (chlordiazepoxide) might be blocked by local injection of a benzodiazepine receptor antagonist (flumazenil) or GABAA receptor antagonist (bicuculline methiodide; BMI) into the region of the anterior basolateral amygdala (BLA) of rats using an ethologically based test of anxiety, the social interaction test. Injection of flumazenil or BMI into the BLA of rats was found to reverse the anxiolytic effects of peripherally administered chlordiazepoxide. These results suggest a major role for the BLA in mediating the anxiolytic effects of benzodiazepines.

Effects of dietary supplementation of vitamin E on storage and caselife properties of lamb retail cuts.

Thirty wether lambs were randomly assigned to three treatments consisting of a control (C) and two vitamin E-supplemented treatments (VE), one fed 500 IU of vitamin E.lamb-1.d-1 (E500) and the other fed 1,000 IU of vitamin E.lamb-1.d-1 (E1000). After a 56-d feeding period, lambs were slaughtered and carcass traits were evaluated. Wholesale legs and loins were vacuum-packaged, stored at 4 degrees C for 7, 14, 21, or 28 d, fabricated into retail cuts, and packaged and displayed to simulate retail industry conditions. The E1000 lambs gained less (P < .05) (kg/d; total gain) and had lower (P < .05) carcass weights than the E500 lambs. Alpha-tocopherol levels in the longissimus lumborum were higher (P < .05) (5.79 vs 3.50 micrograms/g of tissue) for VE than for C; however, there was no difference in alpha-tocopherol level in longissimus lumborum between E500 and E1000. Leg retail cuts experienced greater (P < .05) lipid oxidation and received lower (P < .05) lean color scores than did loin retail cuts. Less (P < .05) lipid oxidation occurred from 1 to 7 d of display in VE retail cuts than in C retail cuts. Longer storage periods before retail display resulted in greater (P < .05) lipid oxidation at both 1 and 7 d of display and a higher (P < .05) rate of lipid oxidation during the display period. Supplementing vitamin E had the greatest effect in reducing lipid oxidation when cuts were stored for longer periods before retail display.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockade of GABAA receptors in the region of the anterior basolateral amygdala of rats elicits increases in heart rate and blood pressure.

Stimulation of the amygdala in rats is known to elicit increases in heart rate (HR) and blood pressure (BP) as well as locomotor activity associated with emotional arousal. The present study was conducted to localize and characterize the role of the GABA system of the amygdala in regulating these cardiovascular responses. Male Sprague-Dawley rats with arterial catheters placed for physiological measurements were implanted with chronic microinjection cannulae in the anterior basolateral (BLA) and central (Ce) amygdaloid nuclei under pentobarbital anesthesia. After recovering, rats were microinjected bilaterally with saline (250 nl) and bicuculline methiodide (BMI, 5-25 ng/250 nl), a selective GABAA antagonist. Microinjection of BMI in the BLA caused significant increases in HR and BP as well as locomotor stimulation while saline had no effect. The cardiovascular response to BMI was blocked by pentobarbital anesthesia. Microinjection of equimolar concentrations of (+)-baclofen HCl (GABAB agonist), phaclofen (GABAB antagonist), or strychnine (glycine antagonist) into the BLA or BMI into the Ce had no significant cardiovascular effects. The cardiovascular effects of BMI injection in the BLA does not appear to be secondary to generalized seizure activity. These results suggest that endogenous GABA, acting on GABAA receptors in the region of the BLA, may be involved in cardiovascular regulation.

Cell-cell adhesion mediated by CD8 and human histocompatibility leukocyte antigen G, a nonclassical major histocompatibility complex class 1 molecule on cytotrophoblasts.

The lymphocyte differentiation marker CD8 acts as a coreceptor with the T cell receptor (TCR) during recognition of peptide presented by major histocompatibility complex (MHC) class I molecules. The functions of CD8 in the TCR complex are thought to be signaling through the association of CD8 with the protein tyrosine kinase p56lck and adhesion to MHC class I through the alpha 3 domain. While the ability of the CD8 alpha/alpha homodimer to bind to classical MHC class I molecules has been shown, it is unclear whether CD8 can also bind nonclassical molecules. Of particular interest is human histocompatibility leukocyte antigen (HLA)-G which is expressed on placental cytotrophoblast cells. These cells do not express HLA-A, -B and -C molecules. In this report, we demonstrate that CD8 can bind to HLA-G. It is possible, therefore, that a cell bearing CD8 may interact with HLA-G-expressing cells.

Mutations in CD8 that affect interactions with HLA class I and monoclonal anti-CD8 antibodies.

The T cell co-receptor, CD8, binds to the alpha 3 domain of HLA class I (Salter, R.D., R.J. Benjamin, P.K. Wesley, S.E. Buxton, T.P.J. Garrett, C. Clayberger, A.M. Krensky, A.M. Norman, D.R. Littman, and P. Parham. 1990. Nature [Lond.]. 345:41; Connolly, J.M., T.A. Potter, E.M. Wormstall, and T.H. Hansen. 1988. J. Exp. Med. 168:325; and Potter, T.A., T.V. Rajan, R.F. Dick II, and J.A. Bluestone. 1989. Nature [Lond.]. 337:73). To identify regions of CD8 that are important for binding to HLA class I, we performed a mutational analysis of the CD8 molecule in the immunoglobulin (Ig)-like variable domain. Our mutational analysis was based on our finding that using a cell-cell adhesion assay murine CD8 (Lyt-2) did not bind to human class I. Since the interaction of human CD8 with HLA class I is species specific, we substituted nonconservative amino acids from mouse CD8 and analyzed the ability of the mutated CD8 molecules expressed in COS 7 cells to bind HLA class I-bearing B lymphoblastoid cells, UC. Mutants with the greatest effect on binding were located in a portion of the molecule homologous to the first and second hypervariable regions of an antibody combining site. In addition, a panel of 12 anti-CD8 monoclonal antibodies were used to stain the 10 CD8 mutants, and amino acids that affected antibody binding were localized on the crystal structure of the Bence-Jones homodimer, REI. Support for an Ig-like structure of CD8 can be found in the pattern of substitutions affecting antibody binding. This work supports the similar tertiary structure of the CD8 alpha-terminal domain and an Ig variable domain.

Biochemical nature of an Fc mu receptor on human B-lineage cells.

An IgM-binding protein of approximately 60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (Fc microR). The Fc microR can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation. Receptor expression is not directly linked with IgM production, as both mu- pre-B cells and isotype-switched B cells may express the Fc microR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with O-linked, but no N-linked, oligosaccharide. The Fc microR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fc microR is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens.

Epstein-Barr virus preferentially induces proliferation of primed B cells.

EBV can induce human B cells to proliferate, differentiate, and undergo transformation into continuously growing lymphoblastoid cell lines. The EBV responsiveness appears to be confined to a very limited subpopulation of B cells, the nature of which is still unclear. In these studies, we sorted tonsillar B cells on the basis of their expression of the early surface activation Ag, Bac-1, and compared their proliferative responses to EBV. Bac-1+ cells responded to EBV with a relatively high level of DNA synthesis, whereas the Bac-1- cells did not. Both large and small Bac-1+ cells were responsive to EBV and the responsiveness was unrelated to the level of Bac-1 immunofluorescence intensity. Bac-1+ cells were relatively enriched for surface IgM and IgD expression. When the Bac-1- population was enriched for IgM+ cells, the proliferative response was still significantly lower than that of the Bac-1+ population. B cells acquire the ability to bind IgM relatively late after activation, and this feature did not distinguish the EBV-responsive B cells. The results suggest B cells become responsive to EBV after an early activation signal.

IgM binding protein expressed by activated B cells.

We have identified an IgM binding protein, a single chain polypeptide of Mr 60,000, that is expressed on the surface of B lymphocytes within 18 hr following their activation with phorbol myristate acetate. The IgM binding protein was also detected on fresh leukemic B cells from individuals with chronic lymphocytic leukemia, and the level of its expression was increased after phorbol myristate acetate activation. Resting and phorbol myristate acetate-activated T cells, monocytes, and polymorphonuclear leucocytes did not express detectable amounts of the IgM binding protein. The 60-kDa protein on activated human B cells could bind secreted IgM molecules of both mouse and human origin, as well as endogenous membrane-bound IgM molecules following their cross-linkage with anti-mu antibodies. The binding of soluble IgM molecules to the surface of activated B cells was also demonstrated by indirect immunofluorescence analysis.

Identification of an early activation antigen (Bac-1) on human B cells.

We have produced a monoclonal antibody, Bac-1, that appears to identify a novel antigen on activated human B cells. The Bac-1 antigen can be detected between 8 to 16 hr, as well as transferrin receptors (T9), after activation of small resting B cells with phorbol myristic acetate, anti-IgM antibody, Staphylococcus aureus Cowan I, or Epstein-Barr virus. The expression of the Bac-1 antigen precedes that of IL 2 receptors (Tac-1). Peak expression of the Bac-1 antigen was observed on day 3 after activation, and decreased thereafter. The Bac-1 antigen was present on a minor subpopulation of relatively large B cells isolated from blood samples, and on "preactivated" B cells of heterogeneous size isolated from spleens and tonsils. It was not detected on bone marrow pre-B cells, blood small B cells, or plasma cells, nor was it expressed by resting or activated T cells or nonlymphoid cells. Certain B cell neoplasms and B lymphoblastoid cell lines were Bac-1+, but neoplastic cells of non-B lineage were Bac-1-. With immunoperoxidase staining, Bac-1+ cells were detected predominantly in the germinal centers of tonsil sections. The Bac-1 antigen on activated B cells was destroyed by protease treatment and was enhanced by neuraminidase treatment, suggesting that the Bac-1 antibody detects a cell surface molecule via an antigenic determinant which is partially obscured by neighboring sialic acid residues. The reactivity pattern of Bac-1 differs from the patterns of cellular reactivity reported for other monoclonal antibodies with specificity for activated human B cells.

A lymphocyte cell surface molecule that is antigenically related to actin.

When viable murine lymphocytes are incubated sequentially with a saturating amount of affinity-purified, rabbit anti-actin and highly conjugated FITC-goat anti-rabbit Ig, about 52% of mesenteric lymph node (MLN) lymphocytes and 36% of thymocytes exhibit a faint, but sharply punctate surface fluorescence. Cell surface actin (CSA) can be distinguished from staining of cytoplasmic actin in permeable cells, which are identified by their uptake of ethidium bromide. Staining of actin in ethidium bromide-permeable cells is 10-fold more intense than staining of actin on ethidium bromide-impermeable cells and is seen as uniformly fluorescent rings or crescents at the periphery of the cell and as dimmer, diffuse fluorescence centrally. Binding of rabbit anti-actin and goat anti-rabbit Ig to the lymphocyte cell surface is not mediated by Fc receptors; F(ab')2 fragments of these antibodies detect the same number of positive cells as do the intact molecules, and affinity-purified anti-KLH does not bind significantly. The cell surface stain, measured by flow cytometry or fluorescence microscopy, can be absorbed by pretreatment of the anti-actin with immobilized actin but not with IgG-Sepharose. Double-label experiments show that about 70% of the non-B cells and 30% of the MLN B cells bear detectable CSA. Although we have not ascertained the origin of CSA, we find that the number and brightness of cells exhibiting CSA cannot be increased by preincubating the cells with exogenous native skeletal muscle actin or with supernatant from dissociated MLN, indicating that there are no free binding sites for exogenous actin. The findings imply that either there is a developmentally expressed binding site(s) for actin, or that at various stages of development lymphocytes express a protein antigenically related to actin on their surface.

F(ab')2 reagents are not required if goat, rather than rabbit, antibodies are used to detect human surface immunoglobulin.

The present report provides evidence that whole goat anti-human immunoglobulin, unlike similar reagents produced in the rabbit, binds both to the same number of and the same individual cells as the F(ab')2 fragments of rabbit or goat anti-human immunoglobulin. These results suggest that goat IgG has a lower affinity for the Fc receptors of human lymphocytes and monocytes than rabbit IgG. Because of this property, whole goat antibodies against human immunoglobulin can be used as simple, convenient relatively inexpensive reagents for the routine detection of immunoglobulin on cell surfaces by immunofluorescence microscopy. The preparation of F(ab')2 fragments of anti-immunoglobulin, which are necessary when rabbit antibodies are used, does not appear to -e required if goat antibodies can be empolyed. This observation has multiple practival applications in cellular immunology.

A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy.

Human leukocytes fixed in suspension were allowed to settle onto poly-L-lysine-coated glass coverslips and prepared for observation with the scanning electron microscope (SEM). The coverslips were dehydrated in ethanol, critical point dried with CO2, and coated with gold-palladium. With the aid of a locator grid, several fields were photographed with light microscopy after the cells had settled onto the poly-L-lysine-coated coverslips and again after completion of the processing before SEM observation. Quantitative comparison of the number of cells present after settling with the number retained for final viewing with the SEM revealed a cell yield approaching 100%. This simple, reproducible, high-yield technique for processing cells fixed in suspension for SEM prevents changes in surface architecture induced by collecting live cells onto various substrates before fixation and also avoids potentially selective cell losses. Such a technique should allow quantitative correlations between SEM and other morphological and functional parameters.