Production of immunoreactive growth hormone by mononuclear leukocytes

In the present study, we evaluated whether mononuclear leukocytes could synthesize and secrete growth hormone (GH) in vitro. By using RNA slot blot analysis, we detected maximum spontaneous levels of specific GH mRNA in the cytoplasm of rat leukocytes after a 4-h incubation. Northern gel analysis demonstrated that the specific leukocyte GH RNA was polyadenylated and had a molecular mass of 1.0 kb. Further studies using immunofluorescence, antibody affinity chromatography, and Sephacryl gel filtration indicate that leukocytes secrete a high molecular weight (greater than 300,000) and a low molecular weight (approximately 22,000) immunoreactive GH (irGH). A substantial amount of the high molecular weight irGH can be converted to the lower molecular weight form after reduction with mercaptoethanol. The irGH appeared to be de novo synthesized because it could be radiolabeled with tritiated amino acids and its production could be blocked by previous incubation of leukocytes with cycloheximide. The replication of Nb2 rat node lymphoma cells was stimulated by affinity-purified human lymphocyte-derived irGH. The growth stimulation was blocked by specific antibodies to hGH. We conclude that lymphocytes produce an irGH that is similar to if not identical to pituitary GH in terms of bioactivity, antigenicity, and molecular weight. The findings demonstrate a potential regulatory loop between the immune and neuroendocrine tissues.     

Nature of leukemia-associated antigenicity of dinitrophenylated human lymphocytes and lymphoblasts

Using dinitrophenylated human lymphocytes and phytohaemagglutinin-stimulated human lymphoblasts as antigens, antibodies were produced in rabbits. The immunological reactivities of the antisera so produced were tested against various types of leukemic cells after absorbing the sera with pooled normal leukocytes. Both the sera showed reactivity with all types of leukemic cells and no specific affinity for lymphoid leukemic cells was seen. This may suggest the presence of some common antigens on all types of leukemic cells or that dinitrophenylation brings about similar changes on all types of normal leukocytes.     

Photoaffinity labeling of plasma membrane receptors for aldosterone from human mononuclear leukocytes.

Non-genomic effects of aldosterone on the sodium-proton-antiport have been shown in human mononuclear leukocytes which could be related to a new aldosterone membrane receptor. In the present paper plasma membranes from human mononuclear leukocytes were covalently photolabeled with a [125I]-aldosterone derivative. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed significant aldosterone binding at a molecular weight of approximately 50000 Dalton which was absent with 1 microM cold aldosterone, but not cortisol in the binding media. The presence of the sulfhydryl agent dithiothreitol did not affect results suggesting the absence of disulfide bridges in the steroid binding domain of the receptor. These data are the first to define the molecular weight of the membrane receptor for aldosterone.     

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.     

Two distinct cDNAs for human IMP dehydrogenase

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.     

Non-induced leukocyte extract reduces HIV replication and TNF secretion

According to UNAIDS, the global HIV/AIDS epidemic increased to 40 million the number of people living with the virus around the world. Dialyzable leukocyte extract obtained by our group is a low molecular weight dialyzable material from peripheral human leukocytes previously in vitro induced with Sendai virus (DLE-ind), and more recently, from non-induced leukocytes (DLE n/i). Previous results have shown the ability of DLE-ind to inhibit HIV in vitro replication in MT4 cell; to reduce TNFalpha secretion, and to delay in vivo progression to AIDS in early stage of HIV infection. In this work we present evidences that DLE n/i also inhibits HIV in vitro replication and reduces TNFalpha secretion in human whole blood like DLE obtained from induced leukocytes. Taking together these results show that both properties of DLE, HIV in vitro inhibition and TNF production modulation, are not dependent on in vitro Sendai virus induction of leukocytes.     

High molecular weight DNA from fixed cytogenetic preparations.

Cell pellets that have been stored in routine clinical cytogenetic fixative were studied for the presence of intact DNA. A method for the isolation of high molecular weight DNA from fixed cytogenetic preparations of human leukocytes, bone marrow, and cell hybrid cultures is presented. DNA preparations from fixed pellets were cleaved with restriction enzymes, transferred to nitrocellulose filters after agarose gel electrophoresis, and hybridized to radiolabeled probes to demonstrate that fixed cell pellets could yield DNA of sufficient quality for Southern blot hybridization analysis. This protocol may be useful for molecular analysis of DNA from fixed cell pellets of patients who are unavailable for additional sampling.     

Mouse serum growth hormone (GH) binding protein has GH receptor extracellular and substituted transmembrane domains

Predicted amino acid sequences for the mouse GH receptor and the related serum GH binding protein were deducted from cDNAs. Two types of cDNA clones were isolated. Both types coded an identical peptide domain with extensive homology to the extracellular domains of the recently cloned human and rabbit GH receptors. However, while one type of clone also encoded regions with homology to the transmembrane and cytoplasmic domains of the human and rabbit GH receptors, the other encoded a short hydrophilic carboxyl-terminal region in place of the transmembrane domain. It is speculated that these two types of clones encode the high and low molecular weight variants of the mouse GH receptor/serum binding proteins, respectively. The low molecular weight variant has been previously found to constitute the majority of the serum GH binding activity in mice. It is proposed that the substitution of the hydrophilic tail for the transmembrane domain may give the low molecular weight variant its soluble nature and account for its presence in serum.     

Heterogeneity of molecules with low molecular weight isolated from media conditioned by human leukocytes and capable of stimulating colony formation by human granulopoietic progenitor cells

Molecules of low molecular weight, able to stimulate colony formation by human granulopoietic cells, were prepared from media conditioned by normal and leukemic human peripheral blood leukocytes. When molecules from these sources were studied, heterogeneity was observed in their ability, after radioiodination, to bind and suicide granulopoietic progenitors, in their radiolabelled tryptic digestion products and in their biological activity as stimulators of granulocyte colonies.     

Flagellar antibody stimulated opsonophagocytosis of Pseudomonas aeruginosa associated with response to either a- or b-type flagellar antigen

Pseudomonas aeruginosa exhibit one of two flagella types: a homogeneous b type, with molecular weight of 53,000, or a heterogeneous a type (subtypes a0, a1, a2, a3, and a4), with molecular weights ranging from 45,000 to 52,000. Pseudomonas aeruginosa flagellar antiserum was shown to promote uptake of radiolabeled bacteria by mouse polymorphonuclear leukocytes. Bacteria were detected directly associated with washed leukocytes and visualized, by electron microscopy, internalized in polymorphonuclear leukocytes. Phagocytosis was specific for the flagella type (a or b) in that homologous flagella serum enhanced uptake three to four times greater than heterologous serum or normal rabbit serum. An a-type antiserum was shown to enhance phagocytosis of four different a-type strains with varying subantigen types, indicating the presence of a common cross-reactive a0 antigen in this flagella type. Phagocytic killing of internalized bacteria was not seen with the addition of only flagellar antiserum.     

Purification and characterization of beta-glucuronidase inhibitor from Mycobacterium tuberculosis

Factors inhibitory to beta-glucuronidase were found in the culture filtrate and in a bacillary extract of Mycobacterium tuberculosis H37Rv grown for 6 weeks on Sauton medium. The inhibitors were purified by ammonium sulfate fractionation, treatment with n-butanol and streptomycin, and chromatography on DEAE-Sepharose CL-6B. Two inhibitors were obtained from the culture filtrate. The molecular weights were estimated to be 25,500 and 15,500 by gel filtration on a Sephadex G-75 column. Three inhibitors were purified from the bacillary extract, two of which were similar to those from the culture filtrate. The molecular weight of the third inhibitor was 21,000. However, the molecular weight of all the denatured inhibitors was 8,600 in the presence of sodium dodecyl sulfate. The inhibitors contained extremely high amounts of glutamic and aspartic acids and had a highly acidic isoelectric point of pH 2.5. The inhibitors acted noncompetitively against beta-glucuronidase of guinea pig origin at an optimal pH 4.5. beta-Glucuronidases from human peripheral leukocytes and beef liver were partially sensitive to the inhibitors; all the other enzymes tested for sensitivity were unaffected by the inhibitors.     

Binding of thyroxine and triiodothyronine in solubilized nuclear extract of human leukocytes.

An in vitro study was carried out of the interaction of thyroidal hormones with leukocytes and with a solubilized extract of the nuclear fraction of human leukocytes. The respective association constants characterising the binding of triiodothyronine (T3) and thyroxine (T4) to whole leukocytes are roughly equal (for T3, KA = 3.1 X 10(11) 1/mol and for T4, KA = 4 X 10(11) 1/mol) but a 0.4 KCl extract of the nuclear fraction exhibits a different affinity to T3 (KA = 2.16 X 10(11) 1/mol) in comparison with T4 (KA = 1.3 X 10(10) 1/mol). In the nuclear extract, both hormones are bound with the affinity higher for T3 than for T4. The soluble nuclear binding protein in human leukocytes had a molecular weight 46 000, was chromatographically homogenous in chromatography on Sepharose 2B and Sephadex G200, and exhibited a longlasting stability at -25 degrees C, without any marked change in the binding affinity to thyroidal hormones.     

Electrodissociation of high molecular weight complexes of interferon alpha during recycling isoelectric focusing

Natural human interferon alpha has been separated by selective ultrafiltration into low molecular weight components and the molecules exceeding 100K daltons. Interferon associated with a higher molecular weight fraction showed partial pH sensitivity and resisted dissociation after treatment with urea, mercaptoethanol, sodium chloride or significant changes in pH. However, interferon activity was released from high molecular weight components during recycling isoelectric focusing. Electrodissociation was carried out in 1% ampholytes for 574 watt-hours. The interferon activity was concentrated in a pH range of 6-6.5, whereas, the majority of proteins were generally found in a more acidic position. The dissociated interferon was neutralized by polyclonal antibody to human interferon alpha (IFN alpha) and showed no presence of pH labile form. A pH sensitivity of high molecular weight interferon (HMW-IFN) may reflect an aggregation phenomenon rather than intrinsic structural differences.     

Eotaxin and the attraction of eosinophils to the asthmatic lung

Eosinophilic leukocytes accumulate in high numbers in the lungs of asthmatic patients, and are believed to be important in the pathogenisis of asthma. A potent eosinophil chemoattractant is produced in the asthmatic lung. This small protein, the chemokine eotaxin, is synthesized by a number of different cell types, and is stimulated by interleukin-4 and interleukin-13, which are produced by T-helper (Th)2 lymphocytes. Low molecular weight compounds have been developed that can block the eotaxin receptor C-C chemokine receptor (CCR)3, and prevent stimulation by eotaxin. This provides the potential for orally available drugs that can prevent eosinophil recruitment into the lung and the associated damage and dysfunction.     

Human endothelial cell cultures: phenotypic modulation by leukocyte interleukins

We report here that soluble factors from activated mononuclear leukocytes have a dramatic effect on cultured endothelial cells. While human umbilical vein endothelial cells grown under standard conditions show a polygonal, epithelial-like morphology, cells exposed to culture media conditioned by lectin-activated human mononuclear leukocytes become extremely elongated and/or send out numerous cytoplasmic processes, assuming a dendritic configuration. This effect cannot be mimicked by exogenous cyclic AMP, is reversible upon interruption of the treatment, and appears specific for endothelial cells, since it has not been observed so far with other cell types. The shape changes are accompanied by a reorganization of the endothelial cell cytoskeleton: actin microfilament bundles tend to be disposed in parallel arrays, while intermediate filaments and microtubules penetrate up to the extremity of the cytoplasmic processes. Colchicine prevents endothelial cell elongation but only slightly impairs the formation of lateral cell processes ("dendritic configuration"). Purified interleukins were tested for their ability to induce these changes of cell shape. Escherichia coli-recombinant human interleukin 2 had no effect, and gamma-interferon only a slight effect on endothelial cell morphology. Interleukin 1 induced moderate cell elongation, while combined treatment with both interleukin 1 and gamma-interferon resulted in shape changes indistinguishable from those elicited by supernatants of activated mononuclear leukocytes. The possible relevance of the observed endothelial cell changes to the reported angiogenic activity of mononuclear cell products is discussed.     

Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.     

Expression of human tissue kallikrein in differentiated HL-60 cells

The kallikrein-kinin system is a mediator of inflammation in humans. In order to elucidate the range of expression of human tissue kallikrein and its substrates, high and low molecular weight kininogen, in inflammatory cells in vitro, we examined their biosynthesis in the HL-60 cell line by RT-PCR and Southern blot analyses. Prominent expression of tissue kallikrein mRNA occurred in untreated promyelocytic cultures as well as in HL-60 cells that were induced to differentiate toward neutrophilic, monocytic, and macrophagic cells. Under the same inducing conditions, kininogen biosynthesis was undetectable at each differentiation state of HL-60 cultures. These results indicate that the myelomonocytic lineage of human leukocytes is a source of tissue kallikrein, which may be secreted as part of the inflammatory process.     

Lactoferrin from canine neutrophils: Isolation and physicochemical and antimicrobial properties

Lactoferrin has been isolated from canine leukocytes for the first time. Lactoferrin was identified by N-terminal amino acid sequence and by capability to capture ferric cations resulting in a complex with absorbance maximum at 460-470 nm. It is demonstrated that canine lactoferrin resembles the human homolog in some physicochemical properties, i.e. molecular weight, carbohydrate presence, and conditions of protein-iron complex dissociation. Bactericidal activity of dog lactoferrin was demonstrated on the gram-negative bacterium Escherichia coli and gram-positive bacterium Listeria monocytogenes. Bactericidal activity of canine lactoferrin is similar to that of human lactoferrin.     

Differing expression patterns and evolution of the rat kininogen gene family

The present investigation using molecular cloning and sequence analysis concerns the examination of the molecular basis for different expression patterns of two types of the rat kininogen genes. We show that the low molecular weight and high molecular weight forms of K kininogens are produced from a single gene through alternative usage of two 3'-coding regions, whereas only the low molecular weight forms of T kininogens are generated as a result of several mutational changes in the high molecular weight-specifying regions of both T-I and T-II kininogen genes. The mutational changes include a nucleotide substitution at the polyadenylation/processing signal site, nucleotide deletions resulting in the frame-shift mutation, and an insertion of the type 2 Alu-equivalent sequence. Because kininogens represent a multifunctional protein comprising the proteinase-inhibitory activity, the kinin moiety, and the clotting activity, these results present evidence indicating the molecular basis for the disappearance of a part of the gene functions. We also show that the K and T kininogen genes as well as the two T kininogen genes are extremely homologous, excluding and including the above mutational changes, respectively. These structural relationships allow us to envisage evolutionary processes for the generation of the rat kininogen gene family, particularly for the disappearance of a part of the gene functions.     

Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5–188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.