Human Leukocyte Migration Inhibitory Factor Research Articles

Specific cellular immunity in acoustic neuroma patients.

An indirect leukocyte migration agarose technique to detect cell-mediated immunity was modified to obtain a specific assay for release of human leukocyte migration inhibitory factor. Acoustic neuroma patients exhibited a significant cellular immune response against acoustic neuroma extract (P less than .01) as well as perilymph from acoustic neuroma patients (P less than .01) when compared to healthy control persons. All 19 patients tested reacted to acoustic neuroma extract. Seven of 21 perilymph samples did not elicit migration inhibition. Crossover determination of antigenicity of two negative and four positive perilymph samples against three patients revealed highly reproducible results, uncorrelated to perilymph concentration of potassium and protein. Flow cytofluorometry did not reveal malignant DNA patterns in 10 acoustic neuromas examined. Immunofluorescence studies did not reveal autoantibodies against acoustic neuromas in sera from 11 patients. The responsible antigen(s), the mechanism of immunization, and the diagnostic implications remain to be determined.

Macrophage migration stimulation factor.

Macrophage migration stimulation factor.

Production of human leukocyte migration inhibitory factor (LIF) by lymphocytes stimulated with phytohemagglutinin: II. Effects of cyclic nucleotides

Pharmacological agents known to modify intracellular levels of adenosine and guanosine 3′,5′-cyclic monophosphate (cAMP and cGMP) were tested for their ability to influence the formation of leukocyte migration inhibitory factor (LIF) by human lymphocytes stimulated by phytohemagglutinin (PHA). The cAMP-elevating agents, papaverine (3 × 10 −5 M) and dibutyryl cAMP (10 −4–10 −6 M), reversibly inhibited LIF production. The effect of dibutyryl cAMP was dose related and could be reversed by simultaneous addition of dibutyryl cGMP. Experiments in which lymphocytes were treated with dibutyryl cAMP at different stages of activation showed that: (i) increased levels of cAMP interfered with LIF synthesis and/or secretion rather than the initial PHA-induced membrane events; and (ii) the period in which the lymphocytes were sensitive to cAMP-induced blocking of LIF formation was limited to the early G 1 phase of the cell cycle; i.e., within 22 hr after mitogen activation. The ability of dibutyryl cAMP and papaverine to abrogate LIF production was not due to a general inhibitory effect of these agents on cellular protein synthesis or secretion. In contrast, both drugs had a weak positive effect on the release of [ 3H]leucine-labeled protein from PHA-activated cells. In spite of the fact that cGMP-elevating agents (10 −4 M dibutyryl cGMP, 10 −5 M carbamylcholine, and 10 −5 M imidazole) increased cellular protein synthesis and secretion in unstimulated mononuclear cells, they did not by themselves trigger LIF production, and they did not inhibit or enhance LIF production by PHA-activated cells.

Production of human leukocyte migration inhibitory factor (LIF) by lymphocytes stimulated with phytohemagglutinin: I. Effect of various metabolic inhibitors

The release of leukocyte migration inhibitory factor (LIF) by human lymphocytes stimulated by pulse exposure to phytohemagglutinin (PHA) was investigated. The activation by PHA occurred very rapidly, since measurable amounts of LIF was produced by cells exposed to PHA for only 15 min at 37°C, and maximum stimulation was reached after 1 hr. Once initiated, release of LIF continued without further stimulation for up to 44 hr. The elaboration of LIF required active lymphocyte metabolism, since low temperature and inhibitors of transcription (1 μg/ml actinomycin D) and translation (5 μg/ml puromycin and 1 μg/ml cycloheximide) prevented the release of LIF. Inhibitors of microfilament function (5 μg/ml cytochalasin B), oxidative phosphorylation (10 −2 M sodium azide), and glycolysis (10 −2 M 2-deoxyglucose) also prevented LIF production, whereas inhibitors of DNA synthesis (25 μg/ml mitomycin C) and microtubule function (3 × 10 −5 M colchicine) were ineffective. The period in which the lymphocyte response was sensitive to cytochalasin B was very limited and centered around the initial stage of activation by PHA.

An indirect assay for canine leukocyte migration inhibitory factor (LIF): Demonstration of its species specificity

An indirect assay in agarose-gel was developed to measure secretion of leukocyte migration inhibitory factor (LIF) by canine lymphocytes. Supernatants from cultures of mitogen- or alloantigen-stimulated canine peripheral blood lymphocytes were assayed for LIF activity using purified granulocytes as indicator cells. Canine LIF activity could be detected provided either autologous or allogeneic canine granulocytes were used as indicator cells. In contrast, canine LIF uniformly failed to inhibit the migration of human granulocytes. Likewise, human LIF inhibited the migration of human but not canine granulocytes in this assay. The data indicated that this species specificity could be due to differences in the structures of LIF receptors on canine and human granulocytes.

Human Leukocyte Migration Inhibitory Factor (LIF)

Recently reported experiments suggest that human leukocyte migration inhibitory factor (LIF) has properties of an esterase and a protease with substrate specificities directed against arginine esters and amides. Also reported previously, the synthetic phosphodiester bis-p-nitrophenyl phosphate (BNPP) but not various phosphomonoesters preserve LIF activity in the presence of the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF). In this paper I demonstrate that guanosine 3'5'-cyclic monophosphoric acid (3',5'-cGMP), a naturally occurring phosphodiester, at concentrations in excess of 10(-5)M also protects LIF against PMSF inactivation. The effect seems specific for the diester bond, its position in the nucleotide, and the guanine base. The possibility that LIF may be a multifunctional or an allosteric enzyme regulated by 3',5'-cGMP is discussed.

Human Leukocyte Migration Inhibitory Factor (LIF)

Previous findings suggesting an esterase and protease nature of human leukocyte migration inhibitory factor (LIF) were extended by testing the ability of different protease and esterase inhibitors to reduce LIF activity. The serine-specific inhibitors phenylmethylsulfonyl fluoride (PMSF) and physostigmine (eserine) markedly reduced LIF activity, whereas the histidine-specific inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) were inactive. That LIF might act as an esterase and a protease was further strengthened by the ability of pralidoxime methansulfonate (2-PAM) to reestablish LIF activity of supernatants previously inactivated by PMSF. Furthermore, the arginine amides N-benzoyl-L-arginine-p-nitroanilide (BApNA) and N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (BPVApNA) were shown to satisfy the substrate specificities of the putative LIF enzyme. Indeed, BPVApNA seemed to possess a particularly strong affinity for LIF, indicating its potential role in an enzymatic LIF assay.

The production of heterologous antibodies to the human lymphokine: Leukocyte migration inhibitory factor (LIF)

Human leukocyte migration inhibitory factor (LIF) produced by concanavalin A-stimulated lymphocytes was partially purified by Sephadex G-100 chromatography and immunosorption of protein contaminants. This material was injected into two rabbits, and the IgG-IgA fractions of the resulting antisera (anti-LIF) neutralized LIF induced by antigen (PPD tuberculin) with as equal efficiency as that of LIF induced by mitogen. Anti-LIF activity was neither removed by absorption with control supernatant or normal human serum nor was it suppressed by absorption with lymphocytes or lymphoblasts. On the other hand, antibodies against human lymphoid cells (ALG) did not reduce LIF activity, indicating the difference between anti-LIF and classical ALG. In support of this, anti-LIF, in contrast to ALG, was not cytotoxic to lymphocytes, and it did not inhibit spontaneous T-rosette formation with sheep erythrocytes. In crossed immunoelectrophoresis using a conventional proteinstaining technique, only three precipitates appeared. None of these contained LIF. However, a protein migrating in the prealbumin region appeared to be specific for lymphocyte stimulation. The nature and significance of this product is unknown.

Human Leukocyte Migration Inhibitory Factor (LIF)

Previously reported experiments suggested that an esterase or a protease, or both, might participate in the expression of human leukocyte migration inhibitory factor (LIF). To clarify this further, a wide variety of simple ester were tested for the ability to protect LIF against inactivation by the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF). alpha-N-benzoyl-L-arginine ethylester (BAEE), a typical trypsin substrate, and bis-p-nitrophenyl phosphate (BNPP), a phosphodiester, were the only esters capable of retaining LIF activity in the presence of PMSF. Agents chemically closely related to these esters were inactive. Moreover, the protection afforded by BAEE and BNPP was the king that would be anticipated if the esters and irreversible inhibitor competed for the same site on LIF. Baee and BNPP also protected against inactivation by di-isopropylfluorophosphate (DFP), another irreversible serine esterase inhibitor. In addition, LIF-treated leukocytes partly escaped migration inhibition in the presence of BAEE and BNPP, respectively. These results indicate that human LIF contains a serine residue necessary for lymphokine activity. It is still not proved, however, that LIF as an enzyme is capable of hydrolyzing BAEE and BNPP, although it seems highly possible. The substrate specificities of a putative LIF enzyme are discussed on the basis of the chemical structure of BAEE and BNPP.