摘要:

AbstractLymphocyte‐endothelial cell interactions are mediated in part by multiple lymphocyte surface adhesion/activation molecules and their cognate ligands. We investigated the surface localization of several of these molecules implicated in T cell adhesion and transen‐ dothelial migration mechanisms to determine if spatial regulation of their distribution contributes to these processes. T lymphocyte suspensions were stained to define distribution, ability to be aggregated into energy‐ dependent caps, and potential cocapping of several adhesion structures. CD2, CD44, L‐selectin (LAM‐1, LECCAM‐1), and CDlla/CD18 (LFA‐1) exhibited uniform distribution on the T cell surface by direct immunofluorescence but formed caps in an energy‐ dependent, and therefore cytoskeletally driven, manner when examined by indirect immunofluorescence. CD2 was shown to syn‐cap (unidirectionally cocap) with GD44 and CDlla/CD18 (LFA‐1), an observation potentially related to functional cooperation among these molecules in T cell activation. T cells were also added to endothelial cell monolayers to assess, in a physiologically relevant context, potential surface molecule reorganization. Lymphocytes co‐cultured with human umbilical vein endothelial cells (HUVEC) underwent a profound shape change, from essentially round cells to polarized cells bearing pseudopodia. Immunofluorescent localization of T cell adhesion/activation molecules using confocal microscopy revealed the redistribution of CD2, CD44, and L‐selectin to the pseudopod. In contrast, CDlla/CD18 remained globally distributed on the cell surface, even in severely deformed cells. Both lymphocyte shape change and membrane molecule redistribution appear to be cell‐cell contact‐dependent phenomena requiring intact, viable endothelial cells. Mechanisms that control these events may be critical to lymphocyte recirculation and inflammation.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.1

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractIn the present study we analyzed expression and biochemical properties of the two recently cloned calcium‐binding proteins MRP8 and MRP14, both members of the S‐100 family, in murine myelomonocytic cells. Expression of MRPs was found to be restricted to granulocytes and distinct stages of macrophage differentiation when compared to the expression of other markers (Mac‐1, F4/80) in transformed macrophage cell lines (Ml, RMB.TG, WEHI.TG, J774A, P388D) and resident and exudate peritoneal and tissue macrophages. Similarly, mRNA and protein levels of MRP8/MRP14 in murine bone marrow cells decreased during culture in L cell‐conditioned medium. Applying a cross‐linking technique, the formation of noncovalently associated heteromeric MRP8/MRP14 complexes of 25, 35, and 48 kd was demonstrated. Our data suggest that MRP8/MRP14 expression and complexation are characteristic for granulocytes and distinct stages of macrophage differentiation in mice. Analysis of the MRP8/MRP14+phenotype of macrophages may provide further insights into the mechanisms underlying macrophage differentiation.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.11

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractInop/opmice, immunohistochemical and electron microscopic techniques were used to examine the effects of theOPmutation on dendritic cell populations in lymphoid tissues and skin. In the thymic medulla, T cell zone of lymph nodes, and splenic white pulp ofop/opmice, numbers of NLDC‐145‐positive dendritic cells were not decreased. Compared to the normal litter‐ mates, numbers of BM8‐positive macrophages were reduced in various tissues of the mutant mice, including the lymphoid tissues. These dendritic cells ofop/opmice expressed la antigens but not F4/80 and BM8 antigens. Ultrastructurally, the dendritic cells developed a tubulo‐ vesicular system typical of interdigitating cells, but they were abnormal in that interdigitation of their cytoplasmic processes was not prominent. In the epidermis of theop/opmice, dendritic cells expressed NLDC‐145, F4/80, la antigens, and adenosine diphosphatase or adenosine triphosphatase activity, and numbers of NLDC‐145‐, la‐, or ADPase‐positive dendritic cells were reduced slightly, but these reductions were not significant statistically. Bir‐ beck granules were detected in most of them electron microscopically. These results indicate that nonlymphoid dendritic cells develop in the lymphoid tissues and skin ofop/opmouse, suggesting that they are differentiated from granulocyte‐macrophage colony‐forming cells or earlier hematopoietic cell precursors.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.19

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe expressions of a membrane form of TNF (m‐TNF) by human alveolar macrophages (AM) and autologous blood monocytes from healthy donors were examined. Upon lipopolysaccharide (LPS) stimulation, AM produced 26‐kDa TNF‐α on their cell surface. We designed a bioassay for measuring m‐TNF in which macrophages were fixed with paraformaldehyde after stimulation for 18 h, then m‐TNF activity was assessed as cytotoxicity of fixed macrophages on L929 cells. This assay was specific to m‐TNF because:1) nosoluble factors were contributed to the cytotoxicity of fixed AM,2)anti‐TNF‐α monoclonal antibody completely neutralized m‐TNF activity, and3)m‐TNF activity was not altered after low‐ pH or high‐salt treatment. On LPS stimulation, AM produced significant amounts of m‐TNF earlier than TNF‐α secretion. AM also expressed significant amounts of m‐TNF when stimulated with other bacterial components and their derivatives. Interleukin (IL)‐4 suppressed both m‐TNF production and TNF‐α secretion. p‐Toluene‐ sulfonyl‐L‐arginine methyl ester (TAME) inhibited specifically TNF‐α secretion and not m‐TNF expression. Although blood monocytes produced small amounts of m‐ TNF, monocyte‐derived macrophages showed enhanced m‐TNF after cultivation with GM‐CSF for 10 days. These findings indicate that m‐TNF is expressed as a step in the TNF‐α producing system, and suggest that m‐TNF may play important roles in exhibition of macrophage function in situ.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.29

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have identified three distinct cell phenotypes with respect to the conditions under which cells became susceptible to TNF‐mediated lysis. These conditions include:1) treatment with the protein synthesis inhibitor, cycloheximide;2)contact with activated macrophages, and3)infection with vaccinia virus. Whereas vaccinia virus‐infected 3T3 cells became sensitive to soluble TNF, F5b cells required contact with activated macrophages. We showed that the “macrophage‐resistant” F5m cells did not become sensitive to TNF or to killing by activated macrophages after infection with vaccinia virus. Therefore, vaccinia infection does not sensitize all cells to TNF. We also determined the pathways of lysis for cells after sensitization. Whereas 3T3, LM929, and F5b cells were killed by the process of necrosis, F5m cells lysis was characterized by the release of low mol wt DNA fragments (apoptosis).

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.37

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractTolerance of macrophages to endotoxin (lipopolysaccharide, LPS) can be induced in vitro by LPS itself. We show here that one of the mechanisms of tolerance to LPS can be mediated via an autocrine process. Continuous exposure to LPS is not required to induce macrophage desensitization. Refractoriness to production of tumor necrosis factor (TNF) in response to LPS can be transferred from tolerant to naive macrophage populations by incubation of the latter with the culture supernatant of the former, in the absence of endotoxin. The active factor present in this macrophage‐desensitizing culture supernatant (MD‐Sup) is more efficiently removed by incubation with tolerant macrophages than by incubation with naive macrophages. The refractoriness elicited by treatment with MD‐Sup is restricted to a decreased TNF response to LPS; interleukin‐1 (IL‐1) and IL‐6 responses are not affected.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.45

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe purpose of these studies was to determine whether triggering murine peritoneal macrophages to a tumoricidal state by lipopolysaccharide (LPS) requires protein‐tyrosine phosphorylation. The LPS‐triggered activation of mouse macrophages to lyse syngeneic B16 melanoma cells was significantly inhibited in a dose‐ dependent manner by the protein‐tyrosine kinase (PTK) inhibitors genistein, herbimycin A, and tyrphostin. Genistein was effective only when added to macrophages prior to or simultaneously with LPS. Genistein potently inhibited the productive interaction of macrophages with LPS but had only a minor effect on the action of interferon‐ γ. The effects of genistein on LPS‐triggered macrophage activation were not due to nonspecific changes in macrophage metabolism or toxicity because genistein did not prevent lysis of tumor cells by activated macrophages, nor did it reduce the capacity of macrophages to phagocytose antibody‐opsonized sheep erythrocytes. Western blot analysis with antiphosphotyrosine monoclonal antibody revealed that incubation of macrophages with LPS produced a rapid increase in tyrosine phosphorylation of several proteins and that the induced phosphorylation could be inhibited by effective concentrations of genistein, herbimycin A, or tyrphostin. Taken together, these data indicate that protein‐tyrosine phosphorylation plays an important role in LPS‐induced tumoricidal activation of macrophages.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.53

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractThe restriction of herpes simplex virus type 1 (HSV‐1) gene expression in nonpermissive murine resident peritoneal macrophages (ResPMO) was analyzed at the level of transcription. Using nuclear run‐on assays, we established that transcription of a representative gene from each HSV‐1 kinetic class was initiated in both ResPMO and permissive Vero cells. Using transient expression assays, we observed that the promoters of two early genes were activated by the immediate‐early protein ICP4 in ResPMO. However, the level of trans‐ activation of these promoters by another immediate‐early protein, ICPO, was markedly reduced in ResPMO. Furthermore, the synergistic trans‐activation normally observed between ICPs4and O in Vero cells was noticeably absent in ResPMO. The data indicate that the block in viral gene expression in ResPMO occurs after initiation of viral gene transcription and involves dysfunction of a viral immediate‐early regulatory protein, ICP0.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.61

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have previously reported the presence, in Jurkat T cells, of outward K+currents and inward currents that have been attributed to Ca2+channels. Here, we have studied the effects of dimethyl 1,4‐dihydro‐ 2,6‐dimethyl‐4‐(2‐nitrophenyl)‐3,5‐pyridine‐dicarboxylate (nifedipine) and 4‐(2,l,3‐benzoxadiazol‐4‐yl)‐l,4‐dihydro‐ 2,6‐dimethyl‐5‐methoxy‐carbonylpyridine‐3‐carboxylate (PN200‐110), two dihydropyridines (DHPs) known to inhibit voltage‐dependent Ca2+channel activity in different types of cells, and two inhibitors of internal Ca2+release (muscle cells), ryanodine and 3,4,5‐trimethoxybenzoic acid 8‐(diethylamino)octyl ester (TMB‐8), on thePhaseo‐ lus vulgarisphytohemagglutinin (PHA)‐dependent responses in Jurkat T lymphocytes. Our results show that nifedipine and PN200‐110 inhibit the PHA‐dependent production of interleukin‐2 except when 12‐0‐ tetradecanoyl‐13‐O‐acetyl phorbol is added to the cultures. Ryanodine and TMB‐8 are not inhibitors. The PHA‐dependent Ca2+response is significantly reduced when the cells are preincubated in the presence of the DHPs. Under these conditions, ryanodine has only a small inhibitory effect and TMB‐8 has no effect. In contrast, only ryanodine (50μM) decreases the PHA‐ dependent cytosolic release of Ca2+when the cells are bathed in a medium containing a low concentration of Ca2+(60 nM). The inhibitory effects of nifedipine and PN200‐110 may result from the binding of these DHPs to specific receptor sites as revealed by studies using [3H]PN200‐110 (KD ‐ 8.5 ± 3.1 nM; 2300 ± 500 apparent binding sites/cell). Photoaffinity labeling studies using [3H]azidopine as a probe showed specific incorporation of label into three glycoproteins of molecular mass (± SD) 170 ± 13, 110 ± 25, and 60 ± 17 kd as analyzed by electrophoresis under reducing conditions.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.66

出版商:Wiley    

年代:1993

数据来源: WILEY

摘要:

AbstractT lymphocytes are activated by a complex series of events, but the mechanisms remain unclear. One uncertainty is the time of receptor‐ligand interaction necessary for commitment to DNA synthesis and proliferation. Although this issue has broad implications for the interpretation of T cell activation data, it remains unresolved. Therefore, we examined the temporal activation requirements of rat splenocytes stimulated with con‐ canavalin A (Con A) by measuring proliferation, as well as interleukin‐2 (IL‐2) production and IL‐2 receptor (IL‐2R) expression. Splenocytes stimulated with various Con A concentrations for 3 h did not incorporate significantly more [3H] thymidine than unstimulated splenocytes. Some increase occurred after 6 h of lectin exposure but maximum proliferation occurred only after the 52‐h stimulation. Furthermore, Con A incubations of 6 h or more were required for significant increases in IL‐2 or IL‐2R. Maximum lymphokine production and receptor expression were observed after the 52‐h stimulation. Thus, activation of some primary lymphocytes required only 6 h of stimulation, but much longer mitogen contact was necessary for maximum recruitment.

ISSN:0741-5400    

DOI:10.1002/jlb.53.1.73

出版商:Wiley    

年代:1993

数据来源: WILEY