Differential inhibition of receptor activation by two mouse monoclonal antibodies specific for the human leukotriene B4 receptor, BLT1.

The inflammatory mediator leukotriene B(4) (LTB(4)) binds to and activates a G-protein-coupled receptor named BLT(1). We have previously produced two monoclonal antibodies, named 7B1 and 14F11, that bind specifically to this receptor. Using a HeLa cell line expressing human BLT(1), we find that both antibodies inhibit LTB(4)-induced calcium release, and activation of a MAP-kinase-sensitive luciferase reporter system. The normal chemotactic movement of polymorphonuclear cells towards higher LTB(4) concentrations was also strongly inhibited by both antibodies. Neither antibody was found to activate BLT(1), and experiments using cyclic peptide fragments of the BLT(1) n-terminal and extracellular loops showed that these antibodies bind only to complex epitopes in the tertiary, membrane bound, conformation of the receptor protein. In ligand binding experiments, 7B1 was found to be a competitive antagonist, while 14F11 was a noncompetitive antagonist that inhibited receptor activation, but not agonist (LTB(4)) binding. 14F11 will be a useful tool for studying the mechanisms of receptor activation.

First-generation monoclonal antibodies identifying the human leukotriene B(4) receptor-1.

The leukotriene B(4) receptor (BLTR) is a seven-transmembrane chemoattractant receptor that is important in pro-inflammatory responses. We have produced the first widely applicable monoclonal antibodies against the human BLTR and confirmed the antibody specificity using flow cytometric analysis of three different cell lines stably expressing the recombinant receptor. The antibodies did not cross-react with the recently cloned second LTB(4) receptor, BLTR2, or the Cys LT1 and Cys LT2 receptors. Functional analysis in combination with two-color flow cytometry showed that the BLTR antibodies bind to cells that are activated by LTB(4). The antibodies were shown to recognize BLTR in cell ELISA and immunocytochemistry. Endogenous expression of BLTR in CD15-positive blood leukocytes and in differentiated HL-60 cells was also demonstrated with the antibodies.

Leukotriene B4 is the functional ligand binding to and activating the cloned chemoattractant receptor, CMKRL1.

We recently described a novel chemoattractant receptor, provisionally named CMKRL1, which has turned out to be the first cloned leukotriene (LT) receptor. Present binding assays using tritiated LTB4 and isolated membranes from COS-7 cells, transiently transfected with cDNA encoding this receptor, yielded a linear Scatchard plot showing expression of only a single, high-affinity receptor population with a mean Kd of 2.1 nM and Bmax of 17.0 pmoles/mg protein. Sham-transfected cells exhibited no specific binding. LTB4 elicited concentration-dependent increases in intracellular calcium measured with Fura-2 in individual CHO cells stably expressing CMKRL1. No response was seen with sham-transfected control cells, or in calcium-free medium which suggests that calcium mainly originates from extracellular sources. The LTB4-induced cellular calcium increment was blocked in the presence of a monoclonal antibody, raised against a synthetic peptide corresponding to the extracellular tail of CMKRL1 and capable of visualizing the receptor by fluorescence immunocytochemistry. Taken together the analyses show that LTB4 is the endogenous ligand for CMKRL1 which is, thus, identical to the LTB4 receptor, designated BLTR according to the NC-IUPHAR nomenclature.

Neurohormonal regulation of histamine and pancreastatin secretion from isolated rat stomach ECL cells.

ECL cells are numerous in the acid-producing part of the rat stomach. They are rich in histamine and pancreastatin, a chromogranin A-derived peptide, and they secrete these products in response to gastrin. We have examined how isolated ECL cells respond to a variety of neuromessengers and peptide hormones. Highly purified (85%) ECL cells were collected from rat stomach using repeated counter-flow elutriation and cultured for 48 h before experiments were conducted. The ECL cells responded to gastrin, sulphated cholecystokinin-8 and to high K+ and Ca2+ with the parallel secretion of histamine and pancreastatin. Glycine-extended gastrin was without effect. Forskolin, an activator of adenylate cyclase, induced secretion, whereas isobutylmethylxanthine, a phosphodiesterase inhibitor, raised the basal release without enhancing the gastrin-evoked stimulation. Maximum stimulation with gastrin resulted in the release of 30% of the secretory products. Numerous neuromessengers and peptide hormones were screened for their ability to stimulate secretion and to inhibit gastrin-stimulated secretion. Pituitary adenylate cyclase activating peptide (PACAP)-27 and -38 stimulated secretion of both histamine and pancreastatin with a potency greater than that of gastrin and with the same efficacy. Related peptides, such as vasoactive intestinal peptide, helodermin and helospectin, stimulated secretion with lower potency. The combination of EC100 gastrin and EC50 PACAP produced a greater response than gastrin alone. None of the other neuropeptides or peptide hormones tested stimulated secretion. Serotonin, adrenaline, noradrenaline and isoprenaline induced moderate secretion at high concentrations. Muscarinic receptor agonists did not stimulate secretion, and histamine and selective histamine receptor agonists and antagonists were without effect. This was the case also with GABA, aspartate and glutamate. Somatostatin and galanin, but none of the other agents tested, inhibited gastrin-stimulated secretion. Our results reveal that not only gastrin but also PACAP is a powerful excitant of the ECL cells, that not only somatostatin, but also galanin can suppress secretion, that muscarinic receptor agonists fail to evoke secretion, and that histamine (and pancreastatin) does not evoke autofeedback inhibition.

Increased expression of cholecystokinin-A receptor mRNA in pancreas and cholecystokinin-B receptor mRNA in oxyntic mucosa after porta-caval shunting in rats.

Porta-caval shunting enhances the trophic effects of cholecystokinin (CCK)-A receptor activation on the pancreas and of CCK-B receptor activation on the ECL cells in the oxyntic mucosa of the rat. The aim of the present study was to study the expression of CCK-A and CCK-B receptor mRNA after porta-caval shunting. Different doses of sulfated CCK-8 (CCK-8s) were administered to porta-caval shunting rats and sham-operated rats, 4 weeks after the operations. The pancreatic wet weight and DNA content were measured and the ECL cells in the oxyntic mucosa were counted after four days of continuous subcutaneous infusion. Total RNA was isolated from pancreas and oxyntic mucosa for Northern blot analysis of CCK-A and CCK-B receptor mRNA. Porta-caval shunting per se did not affect plasma CCK level nor the weight or DNA content of the pancreas, but resulted in increased number of ECL cells despite the fact that the serum gastrin concentration was reduced. The trophic response of the pancreas to low doses of CCK-8s was greater in porta-caval shunted rats than in sham-operated rats. Porta-caval shunted rats displayed an increased CCK-A receptor mRNA concentration in the pancreas (after stimulation with CCK-8s) and an increased CCK-B receptor mRNA concentration in the oxyntic mucosa. In conclusion, the porta-caval shunting-evoked enhancement of the trophic effect of CCK-A receptor activation on the pancreas and of CCK-B receptor activation on the ECL cells is associated with enhanced expression of CCK-A receptor mRNA in the pancreas and of CCK-B receptor mRNA in the oxyntic mucosa.

Depletion of enterochromaffin-like cell histamine increases histidine decarboxylase and chromogranin A mRNA levels in rat stomach by a gastrin-independent mechanism.

BACKGROUND: Gastrin activates histidine decarboxylase (HDC) and increases HDC and chromogranin A (CGA) mRNA levels in histamine-producing enterochromaffin-like (ECL) cells in the rat stomach. We have studied how histamine depletion by subcutaneous infusion of the HDC inhibitor alpha-fluoromethyl-histidine (alpha-FMH) affects how ECL cells respond to hypergastrinemia in terms of HDC and CGA mRNA levels. METHODS: In one experiment rats received alpha-FMH for 24 h. In another experiment rats received alpha-FMH, omeprazole (perorally), or a combination of the two drugs for 10 days. In a third experiment antrectomized rats were treated with alpha-FMH for 48 h. The circulating gastrin level, oxyntic mucosal histamine concentration, HDC activity, and HDC and CGA mRNA levels were determined. RESULTS: alpha-FMH for 24 h increased the HDC and CGA mRNA levels without increasing the serum gastrin concentration. alpha-FMH for 10 days increased the serum gastrin concentration twofold. alpha-FMH + omeprazole resulted in the same serum gastrin concentration as after omeprazole alone (eightfold increase). HDC mRNA levels were higher after alpha-FMH + omeprazole than after omeprazole alone. alpha-FMH alone induced an HDC mRNA level that was similar in magnitude to that observed after omeprazole, although the serum gastrin concentration after alpha-FMH was much lower. In antrectomized rats alpha-FMH increased the HDC and CGA mRNA levels without increasing the serum gastrin concentration. CONCLUSION: ECL-cell histamine depletion will increase mRNA levels for HDC and CGA by a gastrin-independent mechanism, possibly involving abolished histamine autofeedback inhibition.

Heterogeneity of renal carcinoma.

Monoclonal antibodies were used to study the expression of three recently characterized basement membrane components and two carbohydrate antigens in 11 renal-cell carcinomas, using immunohistological and biochemical techniques. The expression of several site-specific kidney antigens in renal-cell carcinoma were studied to determine the origin of the carcinoma and if it is possible further classify this type of carcinoma. Tubulointerstitial nephritis antigen (TIN) and two alpha-chains of type IV collagen, alpha 1 (IV) and alpha 3 (IV) were studied. In addition the expression of carbohydrate antigens Lex and SLex, which also exhibit site-specific distribution were characterized. Lex and SLex antibodies stained the majority of the tumours. TIN was expressed in 9 of 11 tumours, the alpha 1 (IV) chain was present in all 11, and the alpha 3 (IV) chain in two of the 11 tumours. Interestingly, the two alpha 3 (IV)-positive tumours were the same two that were negative for TIN. In normal tissue alpha 3 (IV) is found in distal tubules while TIN is found in proximal tubules. Our results are consistent with earlier observations that the proximal tubule is the origin of most renal-cell carcinomas, but the results also indicate that renal-cell carcinoma may originate from the distal tubule.

Immunocytochemical detection of the carbohydrate antigen, Sialyl Lewis(x), in normal human skin and during irritant contact dermatitis.

Sialys Lewis(x) (SLex) is a ligand for the E-selectin and the interaction of E-selectin on the endothelium and SLex on T cells may be important for T-cell migration into the skin. We investigated the expression of SLex on Langerhans cells (LC) in normal skin and on LC repopulating epidermis deprived of LC due to a preceding irritant contact dermatitis. SLex was visualized by fluorescence and light microscopic immunocytochemistry using the monoclonal antibody, CSLEX-1. The results showed that about 40% of LC in normal epidermis express SLex. In the repopulation phase, most of the epidermal cells were CD1a+/SLex. We suggest that SLex is present on epidermal LC that have recently immigrated from the dermis.

Alpha 1-microglobulin is mitogenic to human peripheral blood lymphocytes. Regulation by both enhancing and suppressive serum factors.

Human alpha 1-microglobulin (alpha 1-m), a 26 kilodalton serum glycoprotein, was found to exert mitogenic effects on human peripheral blood lymphocytes (PBL) in serum-free medium. Purified T cells, but not B cells, responded with proliferation to alpha 1-m, but only in the presence of monocytes. The mitogenic activity could be partially neutralized by a mouse monoclonal antibody against alpha 1-m. The mitogenicity was species-specific, since alpha 1-m homologues from rats, guinea pigs and rabbits had no effect on human PBL. In a previous study, no effect of alpha 1-m was seen on PBL in the presence of 20% serum, and, therefore, we studied the influence of different concentrations of serum on the alpha 1-m-induced mitogenicity. Thus, human serum enhanced the mitogenic effects of alpha 1-m on human PBL at 1% concentration (v/v) and suppressed the effects at 10%. The suppressing effect of serum at 10%, but not the enhancing effect at 1%, seemed to be conserved among several species. To test the effect of serum proteins of different molecular sizes, human autologous serum was separated by gel chromatography on Sephadex G-200 into four fractions. Fractions 1 and 2 (roughly containing proteins larger than 100 kilodaltons) suppressed the mitogenic effects of alpha 1-m, while fractions 3 and 4 enhanced the stimulation by alpha 1-m, at 0.5% and concentrations above. It is concluded that the mitogenic effect of alpha 1-m on lymphocytes is regulated by several serum factors, both enhancing and suppressive, that does not have any proliferative effect of their own. It can be speculated that the balance between enhancing and suppressing co-factors in the blood determines the degree of the stimulation of lymphocytes by alpha 1-m. This is compatible with an immunomodulatory role for alpha 1-m, in spite of its relatively constant plasma levels in health and disease.

Catalase and lipopolysaccharide enhance proliferation in the rat mixed lymphocyte reaction.

Proliferation of rat spleen cells in a mixed lymphocyte culture was amplified fivefold or more in the presence of 2000 units of catalase/ml, as measured by [3H]thymidine incorporation. A similar effect was observed with 1 microgram of lipopolysaccharide (LPS)/ml. Addition of polymyxin B abrogated the promotional effect of LPS, but not that of catalase. These results indicate that the hydrogen peroxide generated by some cells in the rat spleen cell mixed lymphocyte culture suppresses the proliferative response. The demonstration that removal of plastic adherent cells (reducing the percentage of monocytes/macrophages by 75-80%) also results in a 5- to 10-fold increase in a subsequent MLR, indicates that some of the adherent cells may be the producers of hydrogen peroxide, which at higher concentrations suppresses the T-cell proliferation. The enhanced proliferation was not mainly due to increased interleukin 2 (IL-2) production, since the IL-2 concentrations of catalase and LPS-containing cultures were lower than those of control cultures.

Immune functions in melanoma patients during treatment with interferon [HuIFN-alpha (Le)] alone or in combination with cimetidine.

While leukocyte interferon was found therapeutically ineffective in a series of 20 patients with metastatic malignant melanoma, subsequent combination treatment with interferon and cimetidine induced 5 complete and 1 partial tumour remissions. Prior to interferon therapy initiation, regressor patients demonstrated a significantly greater ability to mediate antibody-dependent cellular cytotoxicity than progressor patients and also tended to have higher natural killer-cell activity. These differences were more pronounced following in vitro exposure of effector cells to interferon alone or in combination with cimetidine. During therapy the differences decreased to statistically nonsignificant levels. The number of immunoglobulin producing cells and lymphocyte proliferative responses to Con A were found to increase in both patient groups after interferon therapy initiation; but this augmentation vanished gradually upon combined treatment with cimetidine. A gradual decrease of the number of T lymphocytes and granulocytes was also recorded. None of the demonstrated alterations in the activities of circulating lymphocytes appears to be a relevant correlate to the efficacy of combined therapy compared to interferon alone.

Staphylococcal protein A (SpA) does not induce production of interferon-gamma in human mononuclear blood cells.

Preparations of protein A (SpA) from Staphylococcus aureus and low-molecular-weight SpA induce production of interferon-gamma (IFN-gamma) and are potent mitogens when added to human lymphocytes. The IFN-gamma-inducing and main mitogenic activity of these preparations can be separated from SpA by gel filtration and affinity chromatography. These activities can also be partially inhibited by antiserum to staphylococcal enterotoxin A (SEA) in a specific manner. It is concluded that the IFN-gamma-inducing activity and most of the mitogenic activity of SpA preparations are not attributable to intact or low-molecular-weight fragments of SpA but depend on the presence of SEA and other non-SpA products in the preparations of SpA.