NaCN inhibits the stereospecific depletion of LTB4 by guinea pig polymorphonuclear leukocytes

Caseinate elicited suspension of guinea pig peritoneal PMNs synthesized LTB4, 6t-LTB4, 12-epi-6t-LTB4 and 5HETE after incubations with A23187 and arachidonic acid. Concentrations of LTB4 peaked in 3 minutes and were then rapidly depleted. 6t-LTB4 and 12-epi-6t-LTB4 also peaked in concentrations in 3 min but were depleted slower than LTB4. NaCN inhibited the depletion of LTB4 in a dose dependent fashion without dramatically affecting biosynthesis.     

Tissue differences in vascular permeability induced by leukortriene B4 and prostaglandin E2 in the rat

The activity of synthetic LTB₄ and PGE₂, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with ¹²⁵-I-albumin, ⁵¹Cr-erythrocytes, and ⁵⁷Co-EDTA. The rats were then injected through the carotid artery cannula with LTB₄, PGE₂ or a combination of LTB₄ and PGE₂. Eight minutes later the rats were killed and the activity of ¹²⁵I, ⁵¹Cr, and ⁵⁷Co measured in different organs. Changes in vascular permeability were infered from changes in the ratios of the isotope activities. LTB₄ (15 μg/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB₄, 3 μg/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE₂ (1 μg/kg) enhanced vascular permeability only in the caecum. A combination of LTB₄ (3 μg/kg) and PGE₂ (1 μg/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB₄ than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB₄ induced increases in permeability.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B₄(LTB₄). 6- -LTB₄, 12- -6- -LTB₄, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB₄) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB₄ but 5-HETE formation was impaired. (1-¹⁴C) Arachidonate-labeled platelets exposed to MSU released (1-¹⁴C)-arachidonate. (¹⁴C)-12 HETE, (¹⁴C)-HHT and (¹⁴C)-thromboxane B₂. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB₄.     

Binding of leukotriene B4 and its analogs to human polymorphonuclear leukocyte membrane receptors

LTB₄-induced proinflammatory responses in PMN including chemotaxis, chemokinesis, aggregation and degranulation are thought to be initiated through the binding of LTB₄ to membrane receptors. To explore further the nature of this binding, we have established a receptor binding assay to investigate the structural specificity requirements for agonist binding. Human PMN plasma membrane was enriched by homogenization and discontinuous sucrose density gradient purification. [³H]-LTB₄ binding to the purified membrane was dependent on the concentration of membrane protein and the time of incubation. At 20°C, binding of [³H]-LTB₄ to the membrane receptor was rapid, required 8 to 10 min to reach a steady-state and remained stable for up to 50 min. Equilibrium saturation binding studies showed that [³H]-LTB₄ bound to high affinity (dissociation constant, K_d = 1.5 nM), and low capacity (density, B_max=40 pmol/mg protein) receptor sites. Competition binding studies showed that LTB₄, LTB₄-epimers, 20-OH-LTB₄, 2-nor-LTB₄, 6-trans-epi-LTB₄ and 6-trans-LTB₄, in decreasing order of affinity, bound to the [³H]-LTB₄ receptors. The mean binding affinities (K₁) of these analogs were 2, 34, 58, 80, 1075 and 1275 nM, respectively. Thus, optimal binding to the receptors requires stereospecific 5(S), 12(R) hydroxyl groups, a cis-double bond at C-6, and a full length eicosanoid backbone. The binding affinity and rank-order potency of these analogs correlated with their intrinsic agonistic activities in inducing PMN chemotaxis. These studies have demonstrated the existence of high affinity, stereoselective and specific receptors for LTB₄ in human PMN plasma membrane.     

Opposite effects of adrenalectomy on eicosanoid release in rat peritoneal macrophages and spleen

The effect of adrenalectomy on the formation of cyclo-oxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined and compared with that of the spleen. After isolation, the cells and tissues were incubated with [1-¹⁴C] arachidonic acid and the Ca-ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the macrophages of the controls are 6-keto-PGF_1α, TxB₂ and 12-HETE. One peak represents 5, 12 di HETE. Smaller amounts of PGF_2α, PGE₂, PGD₂, LTB₄ and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of LTB₄, 15-HETE and 12-HETE. The increase in the PG is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, the formation of LTB₄ is considerably increased after adrenalectomy. In the spleen, PGD₂ and 12-HETE are decreased after adrenalectomy.The effect of the macrophages is most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid induced peptide with PlA₂ inhibitory activity in adrenalectomized animals. In the decrease in formation in the spleen, the absence of the permissive effect of glucocorticosteroids on the hormone-induced lipolysis may play a role.     

Structural requirement for the action of leukotriene B4 on the guinea-pig lung:importance of double bond geometry in the 6, 8, 10-triene unit

The action of four synthetic 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acids has been compared to the action of natural leukotriene B₄ (LTB₄) in perfused guinea-pig lung and in the parenchymal strip preparations. Synthetic LTB₄ (Fig. 1) having the 6-$\text{cis}$, 8, 10-$\text{trans}$ triene unit was found to be as powerful as natural LTB₄ both for contracting the parenchymal strip and for releasing prostaglandins and thromboxanes from the perfused lung while three other isomers were inactive. The results indicate that the action of LTB₄ on the lung is highly dependent on the geometry of the conjugated triene.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: Myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, Δ⁶-trans-leukotriene B₄, 12-epi-Δ⁶-leukotriene B₄, ω-hydroxy-leukotriene B₄, ω-carboxy-leukotriene B₄, 15S-hydroxy-eicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to TLB₄ on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The ω-hydroxy-LTB₄ appeared more potent than LTB₄ both for inducing a contraction and for releasing prostanoids whereas the ω-carboxy-LTB₄ was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by PFL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB₄ (and other hydroxy acids) actions on the guinea-pig lungs.     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B₄ (LTB₄) analog, 20-trifluoromethyl LTB₄ (20CF₃−LTB₄), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF₃−LTB₄ was equipotent to LTB₄ as a chemoattractant (EC₅₀, 3 nM), produced 50% of maximal activity of LTB₄, and competed with [H] LTB₄ for binding to intact human neutrophil LTB₄ receptors. In contrast to chemotactic activity, 20CF₃−LTB₄ in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 μM on LTB₄-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB₄, 20CF₃−LTB₄ induced neutrophil desensitization to degranulation by LTB₄. The results indicate that hydrogen atoms at C-20 of LTB₄ are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF₃−LTB₄ is a partial agonist for chemotaxis and an antagonist for degranulation syggests that different LTB₄ receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB₄ can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Corticosteroid suppression of lipoxin A4 and leukotriene B4from alveolar macrophages in severe asthma

Background

An imbalance in the generation of pro-inflammatory leukotrienes, and counter-regulatory lipoxins is present in severe asthma. We measured leukotriene B₄ (LTB₄), and lipoxin A₄ (LXA₄) production by alveolar macrophages (AMs) and studied the impact of corticosteroids.

Methods

AMs obtained by fiberoptic bronchoscopy from 14 non-asthmatics, 12 non-severe and 11 severe asthmatics were stimulated with lipopolysaccharide (LPS,10 μg/ml) with or without dexamethasone (10^-6M). LTB₄ and LXA₄ were measured by enzyme immunoassay.

Results

LXA₄ biosynthesis was decreased from severe asthma AMs compared to non-severe (p < 0.05) and normal subjects (p < 0.001). LXA₄ induced by LPS was highest in normal subjects and lowest in severe asthmatics (p < 0.01). Basal levels of LTB₄ were decreased in severe asthmatics compared to normal subjects (p < 0.05), but not to non-severe asthma. LPS-induced LTB₄ was increased in severe asthma compared to non-severe asthma (p < 0.05). Dexamethasone inhibited LPS-induced LTB₄ and LXA₄, with lesser suppression of LTB₄ in severe asthma patients (p < 0.05). There was a significant correlation between LPS-induced LXA₄ and FEV₁ (% predicted) (r_s = 0.60; p < 0.01).

Conclusions

Decreased LXA₄ and increased LTB₄ generation plus impaired corticosteroid sensitivity of LPS-induced LTB₄ but not of LXA₄ support a role for AMs in establishing a pro-inflammatory balance in severe asthma.     

Leukotriene B4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A2

Leukotriene B₄ (LTB₄) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocyte responses including degranulation, superoxide formation, and chemotaxis. In the present research, stimulation of unprimed polymorphonuclear leukocytes (neutrophils) with LTB₄ results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB₄ concentration of 50–75 nM and peaked at 45 s after stimulation with LTB₄. The transient nature of this release can be attributed, in part, to a fast (<60 s) metabolism of the added LTB₄. Moreover, the inhibition of the reacylation of the released arachidonate with thimerosal results in greater than 4-times as much arachidonate released. Thus, a rapid reacylation of the released arachidonate also contributes to the transient nature of its measured release. Multiple additions of LTB₄, which would be expected to more closely resemble the situation in vivo where the cell may come into contact with an environment where LTB₄ is in near constant supply, yielded a more sustained release of arachidonate. No release of [³H]arachidonate was observed when using [³H]arachidonate-labeled cells. This indicates that the release of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine within the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent with the role of cytosolic phospholipase A₂ (cPLA₂) in the release of arachidonate, potent inhibition of the LTB₄-stimulated release was observed with methylarachidonylfluorophosphonate, an inhibitor of cPLA₂ (IC₅₀ of 1 μM). The bromoenol lactone of the calcium-independent phosphospholipase A₂. failed to affect LTB₄-stimulated release of arachidonate in these cells.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A₄ (actually detected as 6-trans-leukotriene B₄, 12-epi-6-trans-leukotriene B₄, abd 5,6-duhydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-D_R-³H]- or [10-L_S-³H]- labeled arachidonic acid, and 6-trans-LTB₄ and its 12-epimer were analyzed. More than 90% of 10-D_R-hydrogen was lost while about 100% of 10-L_S-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A₄.     

Leukotrienes cause eosinophil emigration into conjunction tissue

The ability of LTB₄, LTC₄, the 5S,6R and 5R,6S LTD₄ stereoisomers, and LTE₄ to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological andl ight microscopy techniques. LTD₄ and LTE₄ demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10ng to 1000ng), while there was only a minimal response to LTC₄. LTB₄ produced marked eosinophil inflitrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB₄. The 5R,6S LTD₄ stereoisomer did not evoke any leukocyte infiltrantion. The SRS-A antagonist, FPL 55712, abolished pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10μg to 1000μg. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Leukotriene B4 receptors as therapeutic targets for ophthalmic diseases

Leukotriene B₄ (LTB₄) is an inflammatory lipid mediator produced from arachidonic acid by multiple reactions catalyzed by two enzymes 5-lipoxygenase (5-LOX) and LTA₄ hydrolase (LTA₄H). The two receptors for LTB₄ have been identified: a high-affinity receptor, BLT1, and a low-affinity receptor, BLT2. Our group identified 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT) as a high-affinity BLT2 ligand. Numerous studies have revealed critical roles for LTB₄ and its receptors in various systemic diseases. Recently, we also reported the roles of LTB₄, BLT1 and BLT2 in the murine ophthalmic disease models of mice including cornea wound, allergic conjunctivitis, and age-related macular degeneration. Moreover, other groups revealed the evidence of the ocular function of LTB₄. In the present review, we introduce the roles of LTB₄ and its receptors both in ophthalmic diseases and systemic inflammatory diseases. LTB₄ and its receptors are putative novel therapeutic targets for systemic and ophthalmic diseases.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B₄ were compared, on a molar basis, with those LTC₄, LTD₄, LTE₄, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD₂, PGE₁, PGF_2α, PGI₂, 6-oxo-PGF_1α, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series.LTB₄ similar to LTC₄ and LTD₄ on GPP, in relation to potency and contractions induced, but differed from LTE₄ in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB₄ produced contractions which were resistant to FPL 55712 (1.9μM) and, when given repeatedly, caused tachyphylaxis in GPP,LTB₄ was considerable more active on GPP than the other substances investigated. Further, PGD₂, PGF_2α and PGI₂ contracted GPP, the order of potency being PGD₂ > PGF_2α ? PGI₂ whereas PGE₁ and PGE₂ relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD₄ displaying the highest activity, LTB₄ was inactive on this tissue. 5-HETE and 6-oxo-PGF_1α were inactive on both GPP and GPISM.On the basis of differential effects of LTB₄ on GPP and GPISM, this assay repressents a simple and selective means to distinguish LTB₄-like materials from other naturally-occuring substances likely to be generated in inflammatory fluids.     

Questionable role of leukotriene B4 in monosodium urate (MSU)-induced synovitis in the dog

Monosodium urate (MSU)-induced synovitis in the dog's stifle (knee joint) is similar to an acute gouty attack in man in which a loss of function of the joint correlates with massive influx of neutrophils and the release of an assortment of inflammatory mediators (e.g. histamine, bradykinin, lysosomal enzymes, complement and eicosanoids) into the synovial space. We found in the urate-induced inflammatory exudates 3 hr post MSU the following: 88 million leukocytes/ml (95% neutrophils) and eicosanoid concentrations of LTB₄, LTC₄, and PGE₂ of < 0.1, 1.4 and 20 ng/ml, respectively. Isotonic saline injected knee joints at 3 hr contained 5 million leukocytes/ml (95% neutrophils) and concentrations of LTB₄, LTC₄, and PGE₂ of < 0.1, 0.7 and 0.2 ng/ml, respectively. Intrasynovial injections of 1 μg LTB₄, 10 μg PGE₂ or the combination of LTB₄ and PGE₂ produced no reduction of paw pressure for up to 3 hr. Leukocyte concentrations measured at 3 hr in joints injected with these arachidonic acids metabolites were similar to saline controls. These results question the role of LTB₄ as a chemotactic and inflammatory mediator in urate-induced synovitis in the dog but confirm the importance of PGE₂ and possibly LTC₄ in this model.     

LTA4-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB4 receptor of human polymorphonuclear leukocytes

5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A₄ (LTA₄) in addition to 5,12-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E,11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA₄ was found to be pH-dependent. After incubation of LTA₄ in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA₄ in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC₅₀ of 250 nM, as compared to values of 3.5 nM for leukotriene B₄ (LTB₄) and >500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB₄ totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB₄. The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB₄ receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB₄ receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB₄.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB₄ (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB₄ and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo- LTB₄. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB₄ was at 10⁻⁷M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB₄ was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at C-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB₄. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta⁶ double bond are all important structural features for chemotactic activity with delta⁶ stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies. The data suggested that they acted at the same receptor since even the less active isomers were able to desensitive the neutrophils to LTB₄.