Rabbit Polymorphonuclear Leukocytes Research Articles

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.

Measurement of opsonophagocytic activity of antibodies specific toNeisseria meningitidis serogroup A capsular polysaccharide-serogroup B outer membrane vesicle conjugate in animal model

Neisseria meningitidis is efficiently phagocytosed by polymorphonuclear leukocytes (PMNS) following opsonization with opsonic antibodies; opsonophagocytosis is the primary mechanism for clearance of meningococci from the host. Thus, in testing meningococcal vaccines, the level of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. Our previous studies demonstrated that the conjugation ofN. meningitidis serogroup A capsular polysaccharide (CPSA) to serogroup B outer membrane vesicle (OMV) could induce a high level of bactericidal antibody response against serogroup A meningococci in animals. The purpose of this study was to evaluate opsonophagocytic activity of the conjugate of CPSA to OMV (CPSA-OMV). In order to evaluate the potential efficacy of CPSA-OMV a flow cytometric opsonophagocytic assay was used. The conjugate and controls were injected intramuscularly into four groups of rabbits with boosters on days 14, 28 and 42 following primary immunization. The rabbits were bled prior to injection and two weeks after each injection. Opsonophagocytic activity of antibodies in hyperimmune sera through rabbit PMNS were measured with flow cytometer, using dihydrorhodamine-123 as a probe. The results indicated that our conjugate could induce a highly significant level of opsonophagocytic activity against serogroup A meningococci after 56 days compared to the control groups (P<0.05). We conclude that this conjugate represents a vaccine candidate against serogroups A and B meningococci after further investigation.

Toll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidin of Staphylococcus aureus

Leukocidin (Luk), an exotoxin of Staphylococcus aureus consisting of LukF and LukS, is a hetero-oligomeric pore-forming cytolytic toxin toward human and rabbit polymorphonuclear leukocytes. However, it is uncertain how Luk affects the host immune response. In the present study, we investigated whether Luk has the ability to stimulate mouse bone marrow-derived myeloid dendritic cells (BM-DCs). LukF activated BM-DCs to generate IL-12p40 mRNA, induce intracellular expression and extracellular secretion of this cytokine and express CD40 on their surface, whereas LukS showed a much lower or marginal ability in the activation of BM-DCs than its counterpart component. Similarly, TNF-α was secreted by BM-DCs upon stimulation with these components. Combined addition of these components did not lead to a further increase in IL-12p40 secretion. IL-12p40 production caused by LukF was completely abrogated in BM-DCs from TLR4-deficient mice similarly to the response to lipopolysaccharide (LPS). Polymixin B did not affect the LukF-induced IL-12p40 production, although the same treatment completely inhibited the LPS-induced response. Boiling significantly inhibited the response caused by LukF, but not by LPS. Finally, in a luciferase reporter assay, LukF induced the activation of NF-κB in HEK293T cells transfected with TLR4, MD2 and CD14, whereas LukS did not show such activity. These results demonstrate that LukF caused the activation of BM-DCs by triggering a TLR4-dependent signaling pathway and suggests that Luk may affect the host inflammatory response as well as show a cytolytic effect on leukocytes.

Bactericidal permeability-increasing protein in host defence against gram-negative bacteria and endotoxin.

The bactericidal permeability-increasing protein (BPI) is a highly conserved host-defence molecule produced and stored by myeloid cells only and a major constituent of the primary granules of human and rabbit polymorphonuclear leukocytes. The c. 50 kDa BPI and a c. 23 kDa bioactive N-terminal fragment are cytotoxic only for Gram-negative bacteria. This target-cell specificity reflects the high affinity (apparent Kd: 1-10 nM) of BPI for the lipid A portion of lipopolysaccharide (LPS or endotoxin). Native and recombinant (r) holo-BPI and the N-terminal fragment (rBPI-23) bind with equal affinity to all forms of isolated LPS examined and inhibit the numerous biological effects of LPS in vitro (including in whole blood ex vivo) as well as in animals. Under the same conditions the antibacterial potencies of holo-BPI and rBPI-23 against Gram-negative bacteria with rough chemotype LPS (whether encapsulated or not) are also the same, but against more resistant smooth chemotype Gram-negative bacteria rBPI-23 is up to 30-fold more potent than holo-BPI. Holo-BPI and rBPI-23 protect a broad range of animals against lethal cytotoxic effects of LPS and in some cases against lethal inoculations with live Gram-negative bacteria.

In vitro and ex vivo antioxidant activities of labetalol on rabbit neutrophil respiratory burst

The beta-adrenoreceptor blocker labetalol has demonstrated important antioxidant properties in vitro that inhibit superoxide anion production during normal leukocyte oxidative metabolism. This study investigated the in vitro and ex vivo effects of labetalol on respiratory burst in rabbit neutrophils. The production of superoxide anions was examined in activated purified rabbit neutrophils after intravenous administration of labetalol (4.0 mg/kg of body weight). At a concentration up to 200 mg/L, labetalol did not demonstrate any cytotoxic effects on neutrophils, as determined by enzyme lactate dehydrogenase activity. In the cell-free system, labetalol demonstrated no significant activity, but in formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated rabbit neutrophils, labetalol demonstrated concentration-dependent antioxidant activity. The in vitro 50% inhibitory concentration (IC50) with the fMLP stimulus was 16.5+/-0.21 mg/L in the rabbit neutrophils and 13.2+/-0.16 mg/L in the human neutrophils. In the fMLP-stimulated rabbit polymorphonuclear leukocytes, labetalol demonstrated its peak inhibitory activity (47%) 3 hours after administration. The mechanism by which labetalol acts in the treatment of hypertension may occur from an interaction in the signaling pathway of protein kinase C activation. The antioxidant properties demonstrated in this mechanism contribute to the drug's antihypertensive action and thus, may reduce the risk of injuries inflicted by reactive oxygen species involved in the pathogenesis of hypertension.

Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes.

Staphylococcal gamma-hemolysin (Hlg), leukocidin (Luk), and Panton-Valentine leukocidin (PVL) are two-component and hetero-oligomeric pore-forming cytolytic toxins (or cytolysin), that were first identified in bacteria. No information on the existence of hetero-oligomeric pore-forming cytolytic toxins in bacteria except for staphylococcal strains is available so far. Hlg (Hlg1 of 34 kDa/Hlg2 of 32 kDa) effectively lyses erythrocytes from human and other mammalian species. Luk (LukF of 34 kDa/LukS of 33 kDa) is cytolytic toward human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes, and PVL (LukF-PV of 34 kDa/LukS-PV of 33 kDa) reveals cytolytic activity with a high cell specificity to leukocytes. Hlg1 is identical to LukF and that the cell specificities of the cytolysins are determined by Hlg2 and LukS. Based on the primary and 3-dimensional structures of the toxin components, Hlg, Luk, and PVL are thought to form a family of proteins. In the first chapter of this article, we describe the molecular basis of the membrane pore-forming nature of Hlg, Luk, and PVL. We also describe a requirement of the phosphorylation of LukS and LukS-PV by protein kinase for their leukocytolytic activity besides their pore formation on human leukocytes.Recently, the assembly mechanism of the LukF and Hlg2 monomers into pore-forming hetero-oligomers of Hlg on human erythrocyte membranes has been clarified for the first time by our study using a single-molecular fluorescence imaging technique. We estimated 11 sequential equilibrium constants for the assembly pathway which includes the beginning with membrane binding of monomers, proceeds through single pore oligomerization, and culminates in the formation of clusters of the pores. In the second chapter of this article, we refer to an assembly mechanism of LukF and Hlg2 on human erythrocytes as well as the roles of the membranes of the target cells in pore formation by Hlg. The LukF, LukS, and Hlg2 proteins are derived from the Hlg locus (hlg), and have been found in 99% of clinical isolates of Staphylococcus aureus. In contrast, LukF-PV and LukS-PV are derived from the PVL locus (pvl) which is distinct from the hlg locus, and only a small percentage of clinically isolated S. aureus strains carries pvl. Recently, we discovered pvl on the genome of lysogenic bacteriophages, psiPVL, and determined the entire gene of the phage. We also demonstrated the phage conversion of S. aureus leading to the production of PVL through the discovery of a PVL-carrying temperate phage, psiSLT, from a clinical isolate of S. aureus. In the third chapter of this article, we discuss genetic analyses of the Hlg, Luk, and PVL genes. We also discuss the current status of knowledge of the genetic organization of PVL-converting phages in order to achieve an understanding of their molecular evolution.

Differential regulation of collagen degradation by rabbit keratocytes and polymorphonuclear leukocytes

Purpose. Both activated keratocytes and infiltrated polymor-phonuclear leukocytes (PMNs) contribute to corneal ulceration by degrading stromal collagen. The regulation of such collagen degradation by inflammatory cytokines was investigated with rabbit keratocytes and PMNs cultured in three-dimensional collagen gels. Methods. Rabbit keratocytes or PMNs were cultured for 24 h in three-dimensional gels of type I collagen in the presence of plasminogen and various concentrations of either interleukin (IL)-1a, IL-6, IL-8, or tumor necrosis factor-a (TNF-a). Degradation of collagen during culture was assessed by measurement of released hydroxyproline. Results. IL-1a increased the amount of collagen degraded by keratocytes or PMNs in a dose-dependent manner, whereas IL-6 had no effect on collagen degradation by either cell type. IL-8 increased the extent of collagen degradation by PMNs but not that by keratocytes, and TNF-a promoted collagen degradation by keratocytes but not that by PMNs. Conclusion. Inflammatory cytokines regulate collagen degradation by rabbit keratocytes and PMNs in culture in a differential manner, and therefore may contribute to the roles of these cells in corneal ulceration.

The discovery of the leukotrienes.

In a study of the transformation of polyunsaturated fatty acids in rabbit polymorphonuclear leukocytes in 1976 it was discovered that arachidonic acid is oxygenated at C-5 (1). Subsequently a number of derivatives, including leukotriene B 4 (LTB 4 ), were identified (2-4). Extension of these studies led in 1979 to the discovery of the pivot epoxide intermediate, LTA 4 (5), and the elucidation of the structure of SRS-A (slow-reacting substance of anaphylaxis) as a group of cysteinyl-leukotrienes, namely LTC 4 , LTD 4 , and LTE 4 (6-10). Earlier work had shown that the prostaglandins are formed by oxygenation and further transformation of arachidonic acid and other polyunsaturated fatty acids (11). The first intermediate in the formation of prostaglandins, the endoperoxide PGG 2 , was isolated and identified in 1974 (12). At the same time we introduced the name cyclooxygenase for the enzyme catalyzing the transformation of arachidonic acid into PGG 2 (12). However, the endoperoxides, PGG 2 and PGH 2 , had some biological effects that could not be explained by their conversion to the known prostaglandins (12). This finding eventually led to the discovery of thromboxane A 2 , an unstable platelet-aggregating and vasoconstrictor substance (13). Subsequent work showed that the endoperoxide can also be converted into a derivative, prostacyclin (PGI 2 ), with opposite biological effects (14). Prostaglandins E 2 and I 2 have strong proinflammatory effects. Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) inhibit the enzyme (cyclooxygenase) responsible for conversion of arachidonic acid into prostaglandins and thromboxanes (15). An induced form of the cyclooxygenase (COX-2) seems to play an important role in inflammation, thus opening the possibility of developing antiinflammatory NSAIDs that lack the side effects of the previous generation of such drugs (16, 17). In 1975 antiinflammatory steroids were proposed to inhibit prostaglandin formation by blocking of the release of the precursor acid from phospholipid stores (18). Since steroids and NSAIDs have significantly different antiinflammatory effects it seemed conceivable that some of these differences might be explained by the formation of proinflammatory derivatives of arachidonic acid by cyclooxygenase-independent reactions. Our studies of the metabolism of arachidonic acid in leukocytes unraveled a new metabolic pathway and led to the discovery of the leukotrienes (8). These compounds play a role in allergy and asthma and have pronounced proinflammatory effects (19, 20).

Assembly of Staphylococcus aureus leukocidin into a pore-forming ring-shaped oligomer on human polymorphonuclear leukocytes and rabbit erythrocytes.

Staphylococcal leukocidin consists of two separate proteins, LukS and LukF, which cooperatively lyse human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes. Here we studied the pore-forming properties of leukocidin and the molecular architecture of the leukocidin pore. (1) Leukocidin caused an efflux of potassium ions from rabbit erythrocytes and swelling of the cells before hemolysis. However, ultimate lysis of the toxin-treated swollen erythrocytes did not occur when polyethylene glycols with hydrodynamic diameters of > or = 2.1 nm were present in the extracellular space. (2) Electron microscopy showed the presence of a ring-shaped structure with outer and inner diameters of 9 and 3 nm, respectively, on leukocidin-treated human polymorphonuclear leukocytes and rabbit erythrocytes. (3) Ring-shaped structures of the same dimensions were isolated from the target cells, and they contained LukS and LukF in a molar ratio of 1:1. (4) A single ring-shaped toxin complex had a molecular size of 205 kDa. These results indicated that LukS and LukF assemble into a ring-shaped oligomer of approximately 200 kDa on the target cells, forming a membrane pore with a functional diameter of approximately 2 nm.

Rabbit polymorphonuclear leukocytes release a factor that causes constriction of the coronary vasculature.

Polymorphonuclear leukocytes (PMN) have been shown to have numerous vasoactive effects, particularly in large artery bioassays. This study shows that rabbit PMN passively release a contractile factor that constricts the coronary vasculature of isolated, Langendorff-perfused rabbit hearts. The mechanism of action of this factor does not involve inhibition of nitric oxide (NO), production of cyclooxygenase metabolites, 5-hydroxytryptamine, or endothelin, or the activation of alpha-adrenoceptors but is a Ca2+-dependent process, because the constriction is inhibited by the Ca2+-channel blocker amlodipine. The activity of this factor is significantly inhibited if it is pretreated with trypsin or heated to 90 degreesC for 10 min, and the active factor is concentrated in the retentate of 100-kDa cutoff centrifuge filters, indicating that the factor is a protein >100 kDa in size. This study shows that rabbit PMN spontaneously release a protein factor that causes constriction of isolated, perfused rabbit hearts by a NO-independent but Ca2+-dependent mechanism.

Precipitated immune complexes of IgM induce the generation of reactive oxygen species by rabbit polymorphonuclear leucocytes.

We report that immune complexes of IgM (ICIgM) antibodies and ovalbumin in the form of a precipitate from the equivalence zone induce the generation of reactive oxygen species by rabbit blood polymorphonuclear leucocytes (PMN), as measured by the chemiluminescence (CL) production in the presence of luminol. The kinetics of CL generation induced by ICIgM is quite different from that induced by precipitated immune complexes of IgG (ICIgG): the maximum rate of CL production for ICIgM occurs around 14 min, whereas for ICIgG it occurs about 5 min after incubation with the cells. Also the triggering of the process requires a higher concentration of ICIgM than of ICIgG. Evidence is presented that these effects are not mediated by interaction of the antigen (ovalbumin) with the cell, since immune precipitates of ovalbumin and the F(ab')2 fragment had no effect. Our observations that precipitated ICIgM can also be an effective stimulus for CL generation and thus for O2- production reveal a new functional capability of PMN. These results may have implications for the understanding of the participation of ICIgM (as well as of ICIgG) in inflammatory reactions mediated by PMN in immune complex diseases, and in the mechanisms of defense against microbes and other non-self agents.

Degranulation enhances release of a stable contractile factor from rabbit polymorphonuclear leukocytes

We investigated the release of a stable contractile factor(s) from rabbit isolated polymorphonuclear leukocytes (PMNs; 10(8) cells/ml) incubated in Tyrode buffer at 37 degrees C. PMNs were untreated, stimulated with N-formylmethionyl-leucyl-phenylalanine (FMLP; 0.1 microM), of degranulated with cytochalasin B (1 microM) in combination with FMLP (0.1 microM). Products from unstimulated PMNs incubated for 60 min caused significantly greater contraction of rabbit isolated aorta (0.56 +/- 0.12 g, n = 8) than did products released from PMNs during a 5-min incubation (0.32 +/- 0.07 g, n = 11, P < 0.05). Stimulation alone did not affect contractile factor release; however, products released from degranulated PMNs caused significantly greater aortic contraction (0.48 +/- 0.08 g, n = 5) than products from nondegranulated PMNs (0.24 +/- 0.04 g, n = 5, P < 0.05) after a 5-min incubation. The contractile activity of PMN-derived products was virtually abolished by heat (90 degrees C, 10 min) or protease (trypsin; 166 U/ml, 5 h) treatment. These findings suggest a PMN-derived protein vasoconstrictor(s) is spontaneously released at a slow rate in vitro and that degranulation can enhance this rate of release. Because PMN degranulation in vivo is associated with inflammation, these results support suggestions that PMN-derived contractile factors may contribute to the impaired blood flow observed during postischemic reperfusion.

Phosphorylation of LukS by protein kinase A is crucial for the LukS-specific function of the staphylococcal leukocidin on human polymorphonuclear leukocytes.

Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 microM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous 32P-H3PO4 into LukS on HPMNLs. However, with LukS and LukF together, 0.5 microM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.

Latrunculin a is a potent inducer of aggregation of polymorphonuclear leukocytes

We have shown that latrunculin A, a toxin from a Red Sea sponge which has striking effects in several phenomena dependent on the cytoskeleton, is a potent inducer of aggregation of rabbit polymorphonuclear leukocytes in a dose- and time-dependent manner. From 12 nM to 60 nM toxin the degree of aggregation (after 8 min) in nearly directly proportional to the toxin concentration; the aggregation effect is energy-dependent from the glycolytic pathway. It was also shown that 120 nM latrunculin A, after 5 min incubation, can reduce to more than half the F-actin percent of the leucocytes. These results may contribute to the study of the relations between the actin cytoskeleton of leukocytes and the process of aggregation which is involved in important physiological functions of these cells.

Enhanced in vitro engulfment of Listeria monocytogenes by rabbit polymorphonuclear leukocytes in the presence of sera from immune rabbits

The role of immune serum in the engulfment of Listeria monocytogenes by polymorphonuclear leukocytes (PMNLs) of rabbits was documented employing an in vitro assay. Three serovarieties of L. monocytogenes, viz. serovars 1 2a , 4a and 4b, were separately incubated with PMNLs of nonimmune rabbit and high titre homologous or heterologous antisera. Normal rabbit serum was used as control. The number of L. monocytogenes per neutrophil was counted in stained smears in each assay and opsonic indices were calculated. Higher opsonic indices, i.e. 2.50, 2.09 and 2.56, for the three strains respectively, were obtained when bacteria were engulfed in the presence of homologous antisera as compared to when the bacterial cells were incubated with heterologous antisera, opsonic indices being in the range of 0.87 to 1.63. These results are indicative of a possible role of specific serum factors in at least the internalization of L. monocytogenes by PMNLs of rabbits and therefore in the host defenses against Listerial infections.

Cholesterol feeding enhances vasoconstrictor effects of products from rabbit polymorphonuclear leukocytes.

We have studied the vasoactive properties of products released from rabbit polymorphonuclear leukocytes (PMNs) before and after short-term (4 and 8 wk) dietary supplementation with 1% cholesterol. Plasma cholesterol levels were similar after 4 and 8 wk of cholesterol diet, whereas gross atherosclerotic lesions were present at 4 wk but significantly more extensive after 8 wk. PMN products from all rabbits caused endothelium-dependent contraction of isolated, control (nonatherosclerotic) rabbit aorta submaximally contracted with phenylephrine. However, both 4 and 8 wk of cholesterol feeding resulted in equivalent contractions by PMN products, which were significantly greater than contractions by control PMNs. Endothelium-dependent contraction (by PMN products) and relaxation (by acetylcholine) were attenuated by 8 wk of cholesterol feeding. PMN products attenuated acetylcholine-induced relaxation of aorta from cholesterol-fed rabbits and of control aorta treated with phenoxybenzamine to reduce muscarinic receptor reserve. We conclude that elevation of plasma cholesterol results in increased release of a PMN product(s) that causes endothelium-dependent constriction.

Effects of subminimal inhibitory concentrations of beta-lactam antibiotics against R-plasmid-bearing bacteria.

The effects of subminimal inhibitory concentrations (sub-MICs) of nine beta-lactam antibiotics [penicillin-G, ampicillin (ABPC), cloxacillin, dicloxacillin, nafcillin, cephalothin, cephradine, cefazolin (CEZ) and cefoxitin] against seven different R-plasmid-bearing gram-negative bacterial strains were tested. Morphological changes, such as filamentous development and lysis, occurred at concentrations considerably lower than the MIC values and the presence of R plasmids did not influence the morphological changes compared with those of Escherichia coli NIHJ, which is non-R-plasmid-bearing. Clear cooperation of ABPC and CEZ with rabbit polymorphonuclear leukocytes PMNs against Pseudomonas aeruginosa and Proteus vulgaris and of CEZ against Salmonella typhimurium was observed, but not against the other strains tested. Therefore, the filamentous bacteria produced as a result of exposure to sub-MICs of antibiotics were not always phagocytosed easily. The ratios between the drug concentrations (microgram/ml) at which the morphological changes began to occur (minimal antibiotic concentration) and the MIC values (microgram/ml) were calculated. A large ratio indicated a wide range of effective concentrations below the MIC value for that antibiotic.

The in vivo and in vitro effects of antibodies against rabbit beta 2-integrins.

Beta 2-integrins play a crucial role in the development of an inflammatory response. In ours study, Abs have been used to investigate the role of individual members of this family of adhesion molecules in both in vivo and in vitro assays. An Ab against rabbit LFA-1 effectively inhibited the adhesion of rabbit polymorphonuclear leukocytes to rabbit endothelial cells in culture and was also effective in blocking cell recruitment to the peritoneum and vascular leakage at dermal sites of inflammation. An Ab that inhibited rabbit complement receptor type 3 function in vitro failed to inhibit cell recruitment to the peritoneum or vascular leakage in response to intradermal FMLP. Histologic studies suggested that the anti-complement receptor type 3 Ab may have modified the cell migration process.

Individual and synergistic effects of rabbit granulocyte proteins on Escherichia coli.

Affinity purification of crude acid extracts of rabbit polymorphonuclear leukocytes using Escherichia coli (J5) as adsorbent yields the bactericidal/permeability-increasing protein (BPI), two 15-kD species (p15s), and the two most potent (cationic) defensin species (neutrophil peptides [NP] -1 and -2). Tested in buffered isotonic medium, the relative antibacterial potency of these proteins against E. coli J5 is BPI (IC50 0.2 nM) > p15A (10 nM) > NP -1 (400 nM). Sublethal doses of p15A or NP-1 can synergize with BPI to decrease the dose required to inhibit the growth of E. coli by up to 50-fold. BPI and p15A display similar features of antibacterial action distinct from defensin NP-1, but NP-1 acts synergistically only with BPI and not with p15A. All aspects of the combined action of BPI and NP-1 resemble those observed with higher concentrations of BPI alone, implying that NP-1 enhances BPI potency. Neither NP-1 nor p15A alter the amount of BPI binding to E. coli but BPI enhances binding of p15A to E. coli, raising the possibility that synergy between these two proteins may occur at least partially at the level of binding. The potent synergistic actions of these proteins can also be demonstrated against serum-resistant clinical isolates of encapsulated E. coli tested in whole blood and plasma ex vivo, suggesting that such combined action may contribute to host defense in vivo.

Structural characterization of BPI-modulating 15 kDa proteins from rabbit polymorphonuclear leukocytes: identification of a novel family of leukocyte proteins.

We have previously described the isolation and initial characterization of 15 kDa protein isoforms (p15s) from rabbit polymorphonuclear leukocytes (PMN) that bind to Escherichia coli and modulate the antibacterial actions of other leukocyte proteins on this gram negative organism. We now report that the p15s differ in primary structure. The cloning and sequencing of two distinct p15 cDNAs from a rabbit bone marrow library reveal that two of the isoforms are closely similar in primary structure differing at only two amino acid positions. The p15 cDNAs encode putative signal sequences suggesting a granule-associated localization for these proteins. Analysis of the derived p15 primary structures reveals homology to two leukocyte proteins: CAP-18, an 18 kD lipopolysaccharide (LPS) binding protein from rabbit PMN and cathelin, an 11 kD cysteine protease inhibitor from porcine leukocytes. This structural similarity suggests the existence of a novel family of low molecular weight leukocyte proteins with potential roles in inflammation.