Lenalidomide enhancement of human T cell functions in human immunodeficiency virus (HIV)-infected and HIV-negative CD4 T lymphocytopenic patients.

Suppressed T cell functions in human immunodeficiency virus (HIV) infection were identified and corrected by lenalidomide in middle-aged HIV-infected patients. Chemotaxis of T cells from HIV-infected men (n = 6, mean 43 years) to sphingosine 1-phosphate (S1P) and CCL21 was significantly lower than that of HIV-negative men (n = 6, mean 41 years), and was enhanced significantly up to control levels by 100 and 1000 nM lenalidomide. Generation of interleukin (IL)-2, but not interferon (IFN)-γ, by T cells of middle-aged HIV-infected men was significantly lower than that for controls and was increased significantly by 10-1000 nM lenalidomide up to a maximum of more than 300%. CD4 and CD8 T cells isolated from healthy middle-aged men and reconstituted in vitro at a low CD4 : CD8 ratio typical of HIV infection had depressed chemotaxis to S1P, but not CCL21, and generation of IL-2, but not IFN-γ. Significant enhancement of chemotaxis to S1P and CCL21 was induced by 100-1000 nM lenalidomide only for normal T cells at a low CD4 : CD8 ratio. T cells from HIV-negative middle-aged CD4 T lymphocytopenic patients (n = 3), with a CD4 : CD8 ratio as low as that of HIV-infected patients, had similarly diminished chemotaxis to S1P and CCL21, and depressed generation of IL-2, but not IFN-γ. Lenalidomide at 30-1000 nM significantly enhanced chemotaxis to S1P and IL-2 generation for T cells from HIV-negative CD4 T lymphocytopenic patients as from HIV-infected patients, with less effect on CCL21-elicited chemotaxis and none for IFN-γ generation. Defects in functions of T cells from middle-aged HIV-infected men are partially attributable to CD4 T lymphocytopenia and are corrected by lenalidomide.

Generation of unique mono-hydroxy-eicosatetraenoic acids from arachidonic acid by human neutrophils.

Incubation of [3H]arachidonic acid with the 17,000-g supernatant from homogenates of human neutrophils in the presence of indomethacin generated the unique metabolites 9-OH-5,7,11,14-eicosatetraenoic acid (9-HETE) and 8-HETE, in addition to 12-HETE, 11-HETE and 5-HETE. The human neutrophil chemotactic activity of the HETE products exhibited a rank-order of potency with 5-HETE greater than 8-HETE = 9-HETE greater than 11-HETE = 12-HETE. The expression of chemokinetic activity as well as chemotactic activity suggested that the endogenous production of these principles may influence the mobility of human neutrophils.

Structural determinants of the eosinophil: chemotactic activity of the acidic tetrapeptides of eosinophil chemotactic factor of anaphylaxis.

The acidic tetrapeptides of ECF-A, Ala/Val-Gly-Ser-Glu, exhibit peak in vitro chemotactic activity for human eosinophils at concentrations of 3 X 10(-8) M to 10(-6) M, and rapidly deactivate eosinophils to homologous and other stimuli at concentrations as low as 10(-10) M. The analogue Leu-Gly-Ser-Glu reaches peak activity at 10(-8)M-10(-7)M, while Phe-Gly-Ser-Glu requires 10(-4)M to elicit a peak response. Although inversion of the order of glycine and serine does not alter the eosinophil chemotactic activity of the tetrapeptides, deletion of glycine increases by 10-fold the concentration required for peak chemotactic activity, indicating the critical nature of the spacing between NH2- and COOH-terminal residues. The substituent COOH-terminal tripeptide, which is only marginally chemotactic, irreversibly suppresses eosinophil chemotactic responsiveness at a concentration 10,000-fold higher than concentrations necessary for deactivation by the intact tetrapeptide. The high concentration of tripeptide required for this cell directed effect, which is assumed to be analogous to deactivation, is attributed to the absence of the NH2-terminal residue which would facilitate effective interaction with the eosinophil. A substituent NH2-terminal tripeptide and amides of the NH2-terminal amino acids, which are devoid of chemotactic and deactivating activities, reversibly inhibit the tetrapeptide stimulus in a dose-response fashion. The additional finding that the NH2-terminal tripeptide protects the eosinophil from deactivation by the intact tetrapeptide confirms that the competitive interaction is stimulus specific.

A neutrophil-immobilizing factor derived from human leukocytes. I. Generation and partial characterization.

A factor has been derived from human leukocytes which irreversibly inhibits the response of human neutrophils to diverse chemotactic stimuli without impairing their viability. It is released by both polymorphonuclear and mononuclear leukocytes during incubation in acidic medium, after endotoxin exposure and subsequent incubation in low potassium medium, and during phagocytosis of particles. It is extractable from both leukocyte types and therefore must be preformed. This chemotactic inhibitor is completely separable from contaminating chemotactic activity in the crude supernatants, has a mol wt of 5000, and is inactivated by digestion with trypsin or chymotrypsin. It has been termed a neutrophil-immobilizing factor because it inhibits neutrophils directly and independently of the chemotactic stimulus, and has relatively little effect on human monocyte chemotaxis.

Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B4. Identification of a subset of high affinity receptors that transduce the chemotactic response.

Human polymorphonuclear (PMN) leukocytes bound [3H]leukotriene B4 ([3H]-LTB4) specifically, as assessed by the displacement of 88% or more of the bound radioactivity by a 15,000-fold higher concentration of nonradioactive LTB4 or by micromolar concentrations of structural isomers of LTB4. The specific binding of [3H]LTB4 by PMN leukocytes was characterized by rapid association and dissociation, and was saturable at 800 nM LTB4. The results of computer analyses of the concentration dependence of binding of [3H]LTB4 were consistent with the expression of two classes of receptors having respective mean affinities of 3.9 X 10(-10) M and 6.1 X 10(-8) M and mean densities of 4.4 X 10(3) and 2.7 X 10(5) per PMN leukocyte. Structural isomers of LTB4 inhibited the binding of [3H]LTB4 to PMN leukocytes at concentrations similar to those required to elicit chemotaxis, while chemotactic peptides did not inhibit binding. PMN leukocytes that were deactivated by prior exposure to LTB4 lost high affinity binding sites selectively and concurrently with a reduction in the chemotactic response to LTB4. Chemotactic deactivation altered, but did not eliminate, the low affinity receptors for LTB4 and reduced only minimally the lysosomal degranulation elicited by LTB4. The high affinity receptors for LTB4 on normal human PMN leukocytes appear to transduce the chemotaxis evoked by LTB4 without substantially modifying lysosomal degranulation.

Novel structural determinants of the human neutrophil chemotactic activity of leukotriene B.

A specific 5(S),12(R)-dihydroxy-eicosa-6,8,10(trans/trans/cis), 14(cis)-tetraenoic acid, designated leukotriene B, is generated by the lipoxygenation and subsequent enzymatic hydration of arachidonic acid in a variety of leukocytes. Leukotriene B elicits a maximal human neutrophil chemotactic response in vitro which is similar in magnitude to those evoked by the chemotactic fragment of the fifth component of complement, C5a, synthetic formyl-methionyl peptides, and 5-hydroxy-eicosatetraenoic acid (5-HETE). The neutrophil chemotactic potency of purified leukotriene B, assessed by the 50% effective concentration of 6 x 10(-9) M, is equivalent to that of C5a, but is up to 100-fold greater than that of 5-HETE and of other natural di-HETE isomers. 5(S),12(R)-di-hydroxy-eicosa-6,8,10(all-trans),14(cis)-tetraenoic acid, which differs from leukotriene B only in having a trans-double bond in place of a cis-double bond in the triene portion of the molecule, and acetyl-leukotriene B are significantly less potent neutrophil chemotactic factors than leukotriene B, which indicates that both the conjugated double bonds and the free hydroxyl-group(s) are functionally critical determinants. The capacity of acetyl-leukotriene B to inhibit competitively and selectively the human neutrophil chemotactic response to equimolar concentrations of leukotriene B suggests the existence of a specific subset of receptors for this potent lipid mediator.

A neutrophil-dependent pathway for the generation of a neutral peptide mediator: partial characterization of components and control by alpha-1-antitrypsin.

A biologically active neutral peptide mediator is cleaved from a plasma protein substrate by an alpha-1-antitrypsin-inhibitable serine protease apparently residing on the membrane of the human neutrophil. The peptide mediator has an approximate mol wt of 1,000, and is distinguished from the kinin peptides by a neutral isoelectric point, susceptibility to inactivation by trypsin as well as chymotrypsin and activity on the isolated, atropinized, and antihistamine-treated guinea pig ileum with relatively little action on the estrous rat uterus. The neutrophil protease is fully inhibitable by DFP, trypsin inhibitors from lima or soy bean, and alpha-1-antitrypsin and is associated with the high mol wt fragments of the neutrophil and not the nuclear, lysosomal, or cytoplasmic subcellular fraction. The substrate has an approximate mol wt of 90,000 and is chromatographically separable from kininogen. The exquisite sensitivity of the neutrophil protease to alpha-1-antitrypsin was established both by inhibition with highly purified alpha-1-antitrypsin and by the inability of the protease to generate detectable neutral peptide in a homozygous (ZZ) alpha-1-antitrypsin-deficient patient without heat inactivation of the residual inhibitor. On the other hand, plasma from a (null) alpha-1-antitrypsin-deficient patient supported neutral peptide generation and revealed an additional factor which inactivated neutral peptide.

Stimulus specificity of the generation of leukotrienes by dog mastocytoma cells.

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.

Modulation of human lymphocyte function by C3a and C3a(70-77).

Human C3a and the synthetic octapeptide C3a (70-77), which retains the activities of an anaphylatoxin, inhibit in a concentration-dependent manner the generation of leukocyte inhibitory factor (LIF) activity by human mononuclear leukocytes and T lymphocytes cultured with the mitogens phytohemagglutinin (PHA) or concanavalin A (Con A) or the antigen streptokinase-streptodornase (SK-SD). The generation of LIF activity was inhibited by 50% by 10(-8) M C3a or C3a(70-77) with PHA or Con A as the stimulus, whereas a more than 10-fold higher concentration of C3a(70-77) than C3a was required to achieve the same level of suppression with SK-SD as the stimulus. Similar concentrations of C3a(70-77) inhibited to the same extent the migration of T lymphocytes stimulated by alpha-thioglycerol of Con A. Neither C3a nor C3a(70-77) altered significantly the uptake of [3H]thymidine by human mononuclear cells exposed to PHA, Con A, or SK-SD. The capacity of C3a(70-77)-Sepharose,m but not Sepharose alone, to adsorb or inactivate mononuclear leukocytes required for the generation of LIF activity established a direct interaction. Analysis of the lymphocytes in the effluent from C3a(70-77)-Sepharose columns, using monoclonal antibodies to surface antigens, showed a selective depletion of the helper/inducer population of lymphocytes. C3a might represent an important mediator of the functionally selective regulation of human T lymphocyte activities by the complement system.

Pertussis toxin inhibition of chemotactic factor-induced calcium mobilization and function in human polymorphonuclear leukocytes.

Chemotactic factors stimulate a rapid increase in the cytosolic concentration of intracellular calcium ions ([Ca2+]in) in human polymorphonuclear leukocytes (PMNL), which may be an event that is critical to the expression of chemotaxis and other PMNL functions. Treatment of PMNL with pertussis toxin catalyzes ADP-ribosylation of a protein similar or identical to the inhibiting regulatory protein of adenylate cyclase, Gi, and suppresses the increase in [Ca2+]in elicited by leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine. Chemotactic migration and lysosomal enzyme release elicited by chemotactic factors were inhibited by pertussis toxin with a concentration-dependence similar to that for inhibition of the increase in [Ca2+]in, without an effect on lysosomal enzyme release induced by the ionophore A23187 and phorbol myristate acetate. Activated pertussis toxin catalyzed the [32P]ADP-ribosylation of a 41 kD protein in homogenates of PMNL. The extent of [32P]ADP-ribosylation of this protein was reduced 59% by pretreatment of intact PMNL with pertussis toxin. Pertussis toxin selectively decreased the number of high-affinity receptors for LTB4 on PMNL by 60% without altering the number or binding properties of the low-affinity subset of receptors. Pertussis toxin modification of a membrane protein of PMNL analogous to Gi thus simultaneously alters chemotactic receptors and attenuates the changes in cytosolic calcium concentration and PMNL function caused by chemotactic factors.

An acidic fibroblast growth factor protein generated by alternate splicing acts like an antagonist.

Polymerase chain reaction amplification of cDNA for acidic fibroblast growth factor in several lines of cultured human cells revealed two forms of mRNA. The novel smaller mRNA lacks the entire second coding exon of the acidic fibroblast growth factor gene, whereas the previously identified mRNA consists of three coding exons. The truncated variant of acidic fibroblast growth factor (aFGF') is only 60 amino acids long with an apparent molecular mass of 6.7 kD on sodium dodecyl sulfate gels in contrast to 18 kD for the full-length acidic fibroblast growth factor. aFGF' elicits only minimal fibroblast proliferation and antagonizes the effects of acidic fibroblast growth factor when added exogenously to or when coexpressed with aFGF in BALB/c/3T3 fibroblasts. Thus, the truncated variant of acidic fibroblast growth factor may provide fibroblasts with a unique mechanism for endogenous regulation of their responses to acidic fibroblast growth factor.

Functional characterization of synthetic leukotriene B and its stereochemical isomers.

Leukotriene B (LTB), a potent lipid chemotactic factor for neutrophils, is 5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid (Fig 1), based upon direct comparison of natural LTB with synthetic 5S,12R-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-di-HETE) stereoisomers in three biological assays. Of the six synthetic stereoisomers evaluated, only the 5S,12R,6,14-cis,8,10-trans compound had chemotactic potency for human neutrophils in vitro that was comparable to that of natural LTB, with a concentration of 3 X 10(9-9) M eliciting a one-half maximum response. In contrast, the racemic mixture of 5R,12R- and 5S,12S-6,10-trans,8,14-cis, the racemic mixture of 5S,12R- and 5R,12S-6,10-trans,8,14-cis, the 5S,12R-6,8-trans,10,14-cis, the 5S,12R-6,8,10-trans,14-cis, and the 5S,12S-6,8,10-trans,14-cis stereoisomers required concentrations of 3 X 10(-7) to 1 X 10(-6) M to elicit comparable responses. Only natural LTB and its synthetic counterpart elicited a local neutrophil infiltration when injected into the skin of the rhesus monkey at 10 ng and 100 ng per site. Natural and synthetic LTB at a concentration of 3 X 10(-8) M each provoked an EC25 contractile response of guinea pig pulmonary parenchymal strips in vitro, whereas the other four tested stereoisomers of 5,12-di-HETE were inactive at this concentration. Structure-function analyses suggest that the neutrophil chemotactic activity depends critically upon the C-1 to C-12 domain, including the stereochemistry of the 6-,8-,and 10-olefinic bonds and the presence of both hydroxyl groups.

Molecular and cellular mechanisms of leukocyte chemotaxis.

The application of modern scientific methods to the study of leukocyte function has begun to reveal the molecular and cytostructural bases of the chemotactic responses of these cells. Leukocyte chemotaxis is initiated by the binding of chemoattractants to distinct plasma membrane receptors; this finding alters transmembrane potential and activates ionic fluxes. The subsequent sequence of metabolic processes leads to a rearrangement of cytoskeletal elements that is manifested by orientation and migration of the cells toward the source of the chemotactic gradient.

Leukotriene C4 transport by the choroid plexus in vitro.

Nanomolar concentrations of peptidoleukotrienes evoke sustained cerebral edema and arterial constriction. Peptidoleukotrienes are thus considered to play an important role in eliciting cerebral edema after cerebral ischemia and vasospasm after subarachnoid hemorrhage. It was hypothesized that the choroid plexus, the locus of the blood-cerebrospinal fluid barrier, might minimize the vasoactivity of locally generated or systemically derived leukotrienes by transporting leukotrienes from cerebrospinal fluid into the blood. Consistent with this hypothesis, leukotriene C4 in vitro was transported into and released from isolated rabbit choroid plexus by a system that was specific, energy-dependent, probenecid-sensitive, and depressed by cold temperatures. The accumulation of leukotriene C4 in the choroid plexus was not dependent on tissue binding or metabolism of leukotriene C4.

Leukotriene B4 produces hyperalgesia that is dependent on polymorphonuclear leukocytes.

Leukotriene B4, at the same intracutaneous doses as bradykinin, reduced the nociceptive threshold in the rat paw. The mechanism of leukotriene B4-induced hyperalgesia was distinguished from that of the hyperalgesia elicited by prostaglandin E2 and bradykinin by its dependence on polymorphonuclear leukocytes and independence of the cyclooxygenation of arachidonic acid.

Constituents of human neutrophils that mediate enhanced adherence to surfaces: purification and identification as acidic proteins of the specific granules.

The endogenous constituents of human neutrophils that enhance the adherence of the neutrophils to surfaces have been isolated from sonicates of purified neutrophils. The predominant adherence-enhancing activity in the neutrophil sonicates cofiltered on Sephadex G-75 with a major peak of chemotactic inhibitory activity and exhibited approximately 30,000 mol wt. Sequential isoelectric focusing and electrophoresis in glycerol gradients of the 30,000-mol wt activities resolved two distinct acidic protein with isoelectric points of 3.6-3.8 and 3.3-3.4 that were designated the neutrophil adherence factor (NAF) I and II, respectively. Glutamic acid and aspartic acid together accounted for a total of 18 and 19% of the amino acids in purified preparations of NAF I and NAF II, respectively, whereas the basic amino acids lysine, arginine, and histidine represented <2 and 3% of the total residues. The preincubation of portions of 2 x 10(6) neutrophils with as little as 6 pmol of NAF I or 9 pmol of NAF II enhanced adherence to plastic petri dishes and inhibited chemotactic migration to a maximal extent, with comparable dose-response relationships for the two effects. Neither of the NAF was cytotoxic, exhibited substantial neutrophil chemotactic or chemokinetic activity, or influenced the phagocytosis of sheep erythrocytes sensitized with immunoglobulin (Ig)G. Analyses of subcellular fractions of neutrophils indicated that the NAF are contained predominantly in the specific granules. These distinctive acidic proteins of the specific granules of human neutrophils represent a new class of endogenous constituents that may regulate the involvement of neutrophils in inflammation.

Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors.

The interaction of human neutrophils adherent to plastic petri dishes with the purified chemotactic factors C5a and kallikrein increased their rate of aerobic glycolysis 25-120% and the activity of their hexose monophosphate shunt (HMPS) 100-600%, reaching a plateau after 2 hr at 37 degrees C. The stimulation of either pathway required a chemotactically active stimulus since neither C5 nor prekallikrein or inactivated kallikrein could enhance metabolic activity. Marked suppression of the neutrophil chemotactic response by preincubation with a chemotactic factor to achieve deactivation, 5 x 10(-7) M diisopropyl fluorophosphate, or the neutrophil immobilizing factor (NIF) did not prevent the stimulation of HMPS activity or glycolysis by chemotactic factors. The metabolic inhibitors iodoacetate and 6-aminonicotinamide at concentrations which blocked enhancement of glycolysis or HMPS activity, respectively, partially suppressed the chemotactic response of neutrophils to the chemotactic factors. The capacity of a chemotactic factor to stimulate glucose metabolism of human neutrophils is associated with a maximal chemotactic response, but this stimulation is not alone sufficient for chemotaxis.

Stimulation by leukotriene D4 of increases in the cytosolic concentration of calcium in dimethylsulfoxide-differentiated HL-60 cells.

The C6-sulfidopeptide leukotrienes C4 (LTC4) and D4 (LTD4) evoked increases in the cytosolic concentration of intracellular calcium ([Ca+2]i) in dimethylsulfoxide-differentiated HL-60 cells, as assessed by the fluorescence of quin-2. The increases in [Ca+2]i reached a peak within 15-90 s, attained 50% of the maximum level at 1.2 nM LTD4 and 60 nM LTC4, were greater in maximal magnitude for LTD4 than LTC4, and subsided in 5-7 min. Flow cytometric evaluation of the LTD4-induced increases in [Ca+2]i, reflected in increases in the fluorescence of intracellular indo-1, revealed that a mean of 77% of differentiated HL-60 cells responded, as contrasted with lesser increases in only 50% of undifferentiated HL-60 cells. The capacity of pretreatment of HL-60 cells with LTD4 to prevent subsequent responses of [Ca+2]i to LTC4 and LTD4, and the finding that the serine-borate inhibitor of conversion of LTC4 to LTD4 suppressed concurrently both LTC4-induced rises in [Ca+2]i and increases in adherence to Sephadex G-25 indicated that the responses of HL-60 cells to LTC4 required conversion to LTD4. That pertussis toxin and a chemical antagonist of LTD4 reduced the [Ca+2]i response suggested a dependence on LTD4 receptors. The LTD4-induced increases in [Ca+2]i were dependent on extracellular calcium and diminished by lanthanum, but not affected by nifedipine nor associated with changes in membrane potential, as measured with the fluorescent probe 3,3'-dipentyloxacarbocyanine. Thus, the increase in [Ca+2]i in HL-60 cells, which is coupled to an increase in adherence, appears to involve LTD4 receptor-specific and voltage-independent calcium channels in the plasma membrane.

Platelet-derived growth factor stimulates mouse 3T3 cell mitogenesis and leukocyte chemotaxis through different structural determinants.

Platelet-derived growth factor (PDGF) stimulates both proliferation of fibroblasts and chemotaxis of leukocytes. In this study we compared the mitogenic and chemotactic activities of native PDGF and reduced PDGF. Reduction of PDGF (Mr = 32,000) to its constituent polypeptides (Mr = 14,000 and 17,000) caused a loss of the ability to stimulate proliferation of Balb/c 3T3 cells. However, reduced PDGF retained virtually all of its activity as a chemotactic agent for human neutrophils and monocytes. A half-maximal chemotactic response to both native and reduced PDGF occurred at a concentration of approximately 0.08 nM for neutrophils and 0.1 nM for monocytes. The maximal chemotactic response to reduced PDGF was at least as great as the maximal response to native PDGF. Both native and reduced PDGF stimulated the release of the lysosomal enzyme, beta-glucosaminidase, from neutrophils with a half-maximal response at less than 0.1 nM. However, the net maximum release of this enzyme by PDGF (and reduced PDGF) was significantly less than that stimulated by a maximal concentration of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. These results indicate that different structural determinants are required for the proliferative response of 3T3 cells to PDGF and for the chemotactic response of leukocytes to PDGF.

Lipoxygenation of arachidonic acid as a source of polymorphonuclear leukocyte chemotactic factors in synovial fluid and tissue in rheumatoid arthritis and spondyloarthritis.

The predominant lipoxygenase products of arachidonic acid were extracted and purified from synovial fluid and sonicates of synovial tissue of patients with rheumatoid arthritis (RA), spondyloarthritis (SA), or a noninflammatory arthropathy (NIA). The concentration of 5(S),12(R)-dihydroxy-6,8,10-(trans/trans/cis)-14-cis-eicosatetraenoic acid (leukotriene B4) in synovial fluid was elevated significantly in patients with RA and a positive latex test for rheumatoid factor (P < 0.05, n = 14) and in patients with SA (P < 0.05, n = 10), compared with that of subjects with NIA (n = 9). The content of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), but not of leukotriene B4, was elevated significantly in synovial tissue of seven patients with RA in comparison with that of four subjects with NIA (P < 0.05). A single intra-articular injection of corticosteroid significantly lowered the synovial fluid level of leukotriene B4 in six patients with RA. These data suggest an involvement of the potent chemotactic factors 5-HETE and leukotriene B4 in human inflammatory disease.