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 esters 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 kind 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.     

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.     

Detection of arginine esterase activity in human follicular fluid.

One major fraction hydrolysing arginine ester and amido derivative (major preparation) was detected and separated from human follicular fluid by Cellulofine GCL-2000 gel filtration. The best ester and amide substrates for this preparation were acetyl-glycyl-lysine methyl ester (Ac-Gly-Lys-Me) and D-valyl-L-leucyl-L-arginine-p-nitroanilide (Val-Leu-Arg-pNA), respectively. The preparation contained plasmin and plasminogen; lysine-Cellulofine adsorption and elution changed the substrate specificity of its esterolytic activity. After treatment by lysine-Cellulofine adsorption and elution, the basic and acidic arginine esterase activities of the major preparation were separated by CM- and DEAE-cellulose adsorption and elution, respectively. The substrate specificity of these two esterolytic enzyme activities differed before and after adsorption of the major preparation to the lysine affinity column.     

Human Leucocyte Migration Inhibitory Factor (LIF)

The cyclic nucleotides guanosine 3',5'-cyclic monophosphoric acid (3',5'-cGMP) and cytidine 2',3'-cyclic monophosphoric acid (2',3'-cCMP) but not cyclic phosphodiesters derived from the bases adenine and uracil preserved LIF activity against the blocking effect of the serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF). Phosphomonoesters derived from guanosine and cytidine as well as 2',3'-cGMP and 3',5'-cCMP were all inactive, indicating specificity for phosphodiester bonds and their respective positions in the two active nucleotides. The protection afforded by 3',5'-cGMP and 2',3'-cCMP was dose dependent. Thus, using 10(-3) M PMSF, 3',5'-cGMP was active at concentrations higher than 10(-5) to 10(-4) M, and 2',3'-cCMP at concentrations higher than 3 X 10(-4) to 10(-3) M. The more pronounced LIF-inhibitory effect obtained by increased concentrations of PMSF could be overcome by raising the levels of the nucleotides, indicating that the interactions between PMSF and the nucleotides with LIF were mutally exclusive. The possibility that 3',5'-cGMP and perhaps 2',3'-cCMP function as modulators of LIF is discussed, and models for the function of this lymphokine are proposed.     

The use of human lymphoid cell line lymphokine preparations (LCL-LK) as working standards in the bioassay of human leucocyte migration inhibition factor (LIF)

The leucocyte migration inhibition test in agarose as described by Clausen (1971) was modified into a statistically designed assay of LIF activity using human polymorphonuclear leucocytes from single blood donors. Individual assays included a laboratory standard of lymphokine with LIF activity prepared from the culture supernatants of the RPMI 1788 human lymphoblastoid cell line (LCL-LK). Analysis of 157 LIF assays revealed simple criteria by which the acceptability of an individual assay could be judged before subjecting it to statistical analysis. The failure of LIF assays to meet these criteria of acceptability was particularly associated with low areas of control polymorph migration in the absence of lymphokine (‘spontaneous migration’).We demonstrate that the statistically designed assay permits the measurement, with precision, of LIF activity in units/ml by reference to a working standard of LCL-LK. We illustrate the use of this assay in the measurement of LIF activity generated by tuberculin-stimulated human peripheral blood lymphocytes.     

Human Leukocyte Migration Inhibitory Factor (LIF)

The activity of leukocyte migration inhibitory factor (LIF) obtained from Sephadex-G-100-chromatographed supernatants of concanavalin-A-stimulated human lymphocytes was suppressed by two synthetic serine esterase and serine protease inhibitors (di-isopropylfluorophosphate (DFP) and phenylmethylfulfonyl fluoride (PMSF)). LIF activity was also reduced by the naturally occurring protease inhibitors soybean trypsin inhibitor and aprotinin. The observed effect of DFP and PMSF was irreversible, since elimination of the inhibitors by dialysis did not restore LIF activity. The effect of PMSF was dose-, time-, and temperature-dependent, and hydrolytic products of PMSF as well as sodium fluoride were inactive in blocking LIF. These results suggest that LIF may act as a serine esterase or a serine protease, or both of these, and that this putative enzyme is present in an activated form in supernatants from mitogen-stimulated mononuclear cells.     

Differential Role of Zn2+ in Antigen- and Mitogen-induced Lymphokine Production

The effect on human lymphokine production in vitro of phenanthroline, a Zn²⁺-chelating agent and an inhibitor of carboxypeptidases A and B, was tested. The elaboration of leucocyte migration inhibitory factor (LIF) by tuberculin-sensitized mononuclear cells stimulated with the specific antigen was reduced in a dose-dependent manner, an effect completely restored by addition of excess Zn²⁺. In contrast, phenanthroline did not affect LIF production by mono-nuclear cells activated nonspecifically by phytohaemagglutinin. It is hypothesized that the presence of a Zn²⁺-dependent molecule, possibly a carboxypeptidase, may be necessary for antigen- but not for mitogen-induced lymphokine production.     

Role of Zn2+ and Other Divalent Metal Ions in Human Lymphokine Production in Vitro

The effects of phenanthroline and of various metal ions on human leucocyte migration inhibitory factor (LIF) production were studied. Previously reported preliminary data showed that phenanthroline, a divalent metal ion chelator, reduced the elaboration of LIF in a dose-dependent manner by specifically but not by non-specifically stimulated lymphocytes. This paper shows that the suppression of LIF production caused by phenanthroline could be entirely reversed by Zn²⁺, Ni²⁺ and, most effectively, by Co²⁺. When a battery of divalent cations were tested for direct inhibitory effects on LIF production, Cd²⁺ and, to a lesser extent, Cu²⁺ were found to be effective. Again, only specifically stimulated cells were susceptible. This profile of responses resembles greatly that seen in experiments with carboxypeptidases, indicating that a carboxypeptidase-like, probably Zn²⁺-dependent enzyme is active during antigen-induced lymphokine production. This metalloenzyme may be derived from activated monocytes/macrophages and, like the lymphocyte-activating factor, exerts its activity in the G₁ phase of the cell cycle.     

Purification and characterization of a novel alkaline serine protease secreted by Vibrio metschnikovii

A novel extracellular alkaline serine protease secreted by Vibrio metschnikovii (V. metschnikovii) ATCC700040 cells was purified by three chromatographic steps and characterized in terms of enzymatic kinetics and substrate specificity. The purified enzyme (named AKP-Vm) was composed of a single polypeptide with an apparent molecular weight of 50 kDa on 12% SDS-polyacrylamide gel in the presence of CuCl?. The optimal temperature and the pH for the enzyme were found to be 37?C and 9.5, respectively. However, the enzyme activity was inhibited by inhibitors such as PMSF and aprotinin. AKP-Vm could hydrolyze a peptide bond at the carboxyl side of the arginine residue, as revealed by its amidolytic activity toward a chromogenic substrate, Boc-Val-Pro-Arg-pNA. The kinetic parameters of the enzyme were as follows: KM=0.91 mM, kcat=0.8 sec?1 and kcat/KM=0.88 mM?1sec?1. AKP-Vm protease could cleave various blood coagulation-associated proteins, including fibrinogen, prothrombin and thrombin. In particular, the enzyme showed powerful fibrinogenolytic and fibrinolytic activities, as it could cleave all major chains of fibrinogen and also digest cross-linked fibrin. The results obtained suggest that AKP-Vm is a novel alkaline serine protease that can actively cleave fibrinogen and cross-linked fibrin.     

Cellular co-operation in the production of human leucocyte inhibitory factor by lymphocyte subpopulations stimulated with antigens and allogeneic cells.

The ability of antigens and allogeneic cells to induce lymphokine synthesis and cellular co-operation in lymphokine production was investigated. Human peripheral blood lymphocytes were separated into T- and B-cell populations by sheep red blood cell rosette formation and centrifugation on Ficoll--Isopaque. The cells were then stimulated with PPD, SK-SD, candida and with allogeneic cells. The presence of leucocyte inhibitory factor (LIF) in the culture supernatants was tested by the agarose migration method. The results indicated that T lymphocytes produced LIF after stimulation with antigens and allogeneic cells. In addition, B cells responded to PPD. Except for PPD the stimulants did not induce significant T-cell LIF production in the absence of monocytes. Only autologous monocytes enhanced LIF synthesis after antigenic stimulation, whereas in mixed lymphocyte cultures allogeneic monocytes were as effective as autologous ones. The monocyte helper effect was mediated by soluble factors in mixed lymphocyte cultures and by soluble factors and direct cell--cell contact in antigen-stimulated cultures. No co-operation between T and B lymphocytes was found. B cells did not enhance LIF production by T cells, nor could T cells induce a B-cell response to antigens or allogeneic cells.     

Production of leucocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes.

Supernatants from human mononuclear cells cultured with PHA inhibited the migration of both human polymorphonuclear leucocytes and guinea-pig peritoneal exudate cells, but not human mononuclear cells. Using ultrafiltration it was shown that these supernatants contained two inhibiting factors, the one with a molecular weight of 15,000-50,000 inhibited only guinea-pig peritoneal exudate cells (MIF), whereas the fraction containing molecules of a size between 50,000 and 75,000 specifically inhibited the migration of polymorphonuclear leucocytes (LIF). The polymorphonuclear leucocyte inhibiting activity was heat labile. It is suggested that the leucocyte migration inhibition test is dependent upon the production of a lymphokine (LIF) which acts specifically on polymorphonuclear leucocytes causing their inhibition of migration.     

Activation of kallikrein-kinin system in human plasma with purified serine protease from Dermatophagoides farinae

An aqueous extract from a mite culture, of Dermatophagoides farinae, activated prekallikrein to kallikrein in normal plasma. Crude protein preparation, obtained by ammonium sulfate precipitation (95% saturation) from the extract, exhibited high activity (0.81 units/mg protein) towards a synthetic substrate of coagulation factor XIIa, Boc-Gln-Gly-Arg-MCA, and had also activity to form kallikrein in human plasma deficient in coagulation factor XII. Treatment of the protein preparation with phenylmethylsulfonyl fluoride (PMSF), an inhibitor of serine enzyme, gave rise to inactivation of both activities. Thus, the serine protease specific for Boc-Gln-Gly-Arg-MCA in mite cultures of D. farinae was purified by ammonium sulfate precipitation and chromatographies on p-aminobenzamidine-sepharose CL-4B, DEAE-Toyopearl 650M, Sephadex G-75 superfine and Sephacryl S-200. The purified protease was homogeneous electrophoretically, and its molecular weight was estimated to be 30,000. The optimum pH and temperature were around 7.5 and 50 degrees C, respectively. The specific activity was 36 units/mg protein at pH 7.4 and 37 degrees C. The activity was completely inhibited by PMSF. The serine protease had the activity to activate the kallikrein-kinin system in normal human plasma.     

Affinity chromatography. A new technique for partial purification of human leucocyte migration inhibitory factor

Human leucocyte migration inhibitory factor (LIF) was subjected to affinity column chromatography on Sepharose-bound α-L-fucose. Following the addition of α-L-fucose to the running buffer adsorbed lymphokine was eluted almost quantitatively. A minimum of 70-fold purification of LIF was achieved.     

Demonstration of leukocyte inhibitory factor in human Schistosomiasis mansoni and its evaluation from patients in an endemic setting

Leukocyte migration inhibitory Factor (LIF) is produced by the peripheral blood mononuclear cells (PBMN) of most patients with Schistosoma mansoni infection upon their exposure to soluble egg antigens (SEA). PBMN of some patients also respond to adult worm (SWAP) antigens by LIF production. LIF is stable at 37 degrees C for 60 min but is sensitive to heating at 56 degrees C for even 30 min. The serine protease inhibitor PMSF destroyed LIF activity at concentrations of 10(-2) to 10(-3) M. Concanavalin A stimulated production of detectable levels of LIF by 8 hr, while SEA and SWAP did so by 15 and 39 hr, respectively. PBMN of healthy normal controls did not produce LIF upon exposure to SEA or SWAP. PBMN of a few field controls (stool negative subjects from an endemic area) produced detectable LIF activity when exposed to SEA or SWAP. PBMN from most infected (stool positive) patients from an endemic area produced LIF when exposed to SEA and only occasionally did so to SWAP. Previous studies have shown that most often only the PBMN of former, cured patients, and not chronically infected patients, produce the lymphokine activity termed mitogenic factor (MF). The current data indicate that because LIF is primarily produced by actively infected patients, its production may be controlled by different immunoregulatory mechanisms. Furthermore, although most SEA-related responses are highly immunoregulated in active, chronically infected patients, SEA appears to be a better stimulus for patient PBMN production of LIF than SWAP.     

Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats.

A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin agarose and gel filtration. The apparent molecular mass of the enzyme was 31.5 kDa and the pI was 11.8. The enzyme was not glycosylated and its first 15 N-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 while inactivation of the enzyme was reversible at pH 4, but not at pH 11. The enzyme was stable at 37 degrees C with an apparent optimum temperature around 55 degrees C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0.37 mM) and physiological efficiency (k(cat)/Km) were obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide. These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins G prepared against the keratinase and used in an immunohistochemical test allowed the detection of the keratinase produced by the fungus invading hair structures in naturally infected cats. The in vitro keratinolytic activity of the enzyme and its production in vivo suggest that it may contribute to pathogenicity.     

Characterisation of an alkaline peptidase of Trypanosoma cruzi and other trypanosomatids

A peptidase activity was purified from extracts of Trypanosoma cruzi on the basis of its ability to cleave benzoyl-arginine-p-nitroanilide. The enzyme was considered to be a cysteine-type peptidase with unusually low sensitivity to E-64. It has a pH optimum of about 8.0 for p-nitroanilides, and cleaves peptide bonds on the carboxyl side of arginine and, to a lesser extent, lysine residues. Cleavage of different substrates occurred at rates that were determined by their catalytic constants (kcat): the peptidase had the same Michaelis constant (about 30 microM) for all substrates tested. Evidence is presented that the peptidase is the major cysteine peptidase in T. cruzi extracts that cleaves p-nitroanilides next to basic amino acid residues at pH 8. The enzyme was detected in all stages of the life cycle of T. cruzi. Furthermore, evidence is presented, based on pH optima, inhibitor sensitivity, substrate specificity and kinetics, and electrophoretic mobility, that a similar or identical enzyme occurs in fifteen other species of trypanosomatid.     

A comparative study of two lipases from different strains of Staphylococcus aureus

In order to compare lipases from two different Staphylococcus aureus strains (FN 37 and TEN 5), the enzymes from the respective strains were purified using octyl-Sepharose chromatography and characterized with regard to chemical, immunological and enzymatic properties. Differences in the size of the lipases in their native forms necessitated modifications of the purification process, but after purification identical subunits of about 43 kD were found in SDS-PAGE, and both lipases had an apparent molecular weight of 110 kD when subjected to gel chromatography on Sephadex G-200. Analysis of the amino acid compositions of the lipases showed considerable differences. Serine was the predominant amino acid in the FN 37 lipase, whereas glycine was most abundant in the TEN 5 lipase. Nevertheless, the two lipases were similar when tested with double immune diffusion against an antiserum raised against the TEN 5 lipase, and inactivation of enzyme activities by the antiserum followed the same patterns. Enzymatically, the enzymes were similar with regard to salt inhibition, ion dependency and heat inactivation. The substrate specificities, tested against glyceride substrates, were similar. Thus, the two staphylococcal lipases seem to be similar but not identical.     

Lack of Evidence for Human Lymphokines with Thrombocyte Aggregating Activity

The ability of human lymphocytes to produce soluble mediator(s) with thrombocyte aggregating activity (TAA) was investigated in an in vitro technique. Isolated human mononuclear cells were stimulated with phytohaemagglutinin or purified protein derivative of tuberculin. The supernatants were investigated for lymphokine activity in a leucocyte migration inhibitory factor assay. Supernatants were then tested for their ability to aggregate human thrombocytes, and in contrast to the results of previous studies, we were not able to demonstrate any TAA. Most likely, lymphokines with TAA are not involved in the thrombotic processes seen in human cell-mediated immune inflammatory reactions.     

Arginine esterase activity of the plasma in different types of bronchial asthma

Spontaneous arginine esterase activity and esterolytic activity of plasma after a contact with kaolin, using a synthetic substrate BAEE in asthmatic patients were studied. Also activity of esterase inhibitors, the plasma kininogen level and fibrinolytic activity were determined. It was shown, that during an asthma attack a significant rise in the plasma esterolytic activity occurred. At the same time esterase activity displayed a fall after contact with kaolin as compared with controls. These changes were accompanied by a rise in the fibrinolytic activity of the plasma and a decrease in the kininogen level. The behavior of parameters studies was analyzed in relation to the immunological type of asthma and the clinical state of the patients.