Inflammatory Peritoneal Exudate Research Articles

Sex Hormone-Dependent Lipid Mediator Formation in Male and Female Mice During Peritonitis.

Introduction: Sex differences in inflammation are obvious and contribute to divergences in the incidence and severity of inflammation-related diseases that frequently preponderate in women. Lipid mediators (LMs), mainly produced by lipoxygenase (LOX) and cyclooxygenase (COX) pathways from polyunsaturated fatty acids (PUFAs), regulate all stages of inflammation. Experimental and clinical studies revealed sex divergences for selected LM pathways without covering the entire LM spectrum, and only few studies have addressed the respective role of sex hormones. Here, we performed the comprehensive LM profile analysis with inflammatory peritoneal exudates and plasma from male and female mice in zymosan-induced peritonitis to identify the potential sex differences in LM biosynthesis during the inflammatory response. We also addressed the impact of sex hormones by employing gonadectomy. Methods: Adult male and female CD1 mice received intraperitoneal injection of zymosan to induce peritonitis, a well-established experimental model of acute, self-resolving inflammation. Mice were gonadectomized 5 weeks prior to peritonitis induction. Peritoneal exudates and plasma were taken at 4 (peak of inflammation) and 24 h (onset of resolution) post zymosan and subjected to UPLC–MS-MS–based LM signature profiling; exudates were analyzed for LM biosynthetic proteins by Western blot; and plasma was analyzed for cytokines by ELISA. Results: Pro-inflammatory COX and 5-LOX products predominated in the peritoneum of males at 4 and 24 h post-zymosan, respectively, with slightly higher 12/15-LOX products in males after 24 h. Amounts of COX-2, 5-LOX/FLAP, and 15-LOX-1 were similar in exudates of males and females. In plasma of males, only moderate elevation of these LMs was apparent. At 4 h post-zymosan, gonadectomy strongly elevated 12/15-LOX products in the exudates of males, while in females, free PUFA and LOX products were rather impaired. In plasma, gonadectomy impaired most LMs in both sexes at 4 h with rather up-regulatory effects at 24 h. Finally, elevated 15-LOX-1 protein was evident in exudates of males at 24 h which was impaired by orchiectomy without the striking impact of gonadectomy on other enzymes in both sexes. Conclusions: Our results reveal obvious sex differences and roles of sex hormones in LM biosynthetic networks in acute self-resolving inflammation in mice, with several preponderances in males that appear under the control of androgens.

Non-Myeloid Cells are Major Contributors to Innate Immune Responses via Production of Monocyte Chemoattractant Protein-1/CCL2.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 is a chemokine regulating the recruitment of monocytes into sites of inflammation and cancer. MCP-1 can be produced by a variety of cell types, such as macrophages, neutrophils, fibroblasts, endothelial cells, and epithelial cells. Notably, macrophages produce high levels of MCP-1 in response to proinflammatory stimuli in vitro, leading to the hypothesis that macrophages are the major source of MCP-1 during inflammatory responses in vivo. In stark contrast to the hypothesis, however, there was no significant reduction in MCP-1 protein or the number of infiltrating macrophages in the peritoneal inflammatory exudates of myeloid cell-specific MCP-1-deficient mice in response to i.p injection of thioglycollate or zymosan A. Furthermore, injection of LPS into skin air pouch also had no effect on local MCP-1 production in myeloid-specific MCP-1-deficient mice. Finally, myeloid-specific MCP-1-deficiency did not reduce MCP-1 mRNA expression or macrophage infiltration in LPS-induced lung injury. These results indicate that non-myeloid cells, in response to a variety of stimulants, play a previously unappreciated role in innate immune responses as the primary source of MCP-1.

Tyrosine Phosphorylation of CD13 Regulates Inflammatory Cell–Cell Adhesion and Monocyte Trafficking

CD13 is a large cell surface peptidase expressed on the monocytes and activated endothelial cells that is important for homing to and resolving the damaged tissue at sites of injury. We showed previously that cross-linking of human monocytic CD13 with activating Abs induces strong adhesion to endothelial cells in a tyrosine kinase- and microtubule-dependent manner. In the current study, we examined the molecular mechanisms underlying these observations in vitro and in vivo. We found that cross-linking of CD13 on U937 monocytic cells induced phosphorylation of a number of proteins, including Src, FAK, and ERK, and inhibition of these abrogated CD13-dependent adhesion. We found that CD13 itself was phosphorylated in a Src-dependent manner, which was an unexpected finding because its 7-aa cytoplasmic tail was assumed to be inert. Furthermore, CD13 was constitutively associated with the scaffolding protein IQGAP1, and CD13 cross-linking induced complex formation with the actin-binding protein α-actinin, linking membrane-bound CD13 to the cytoskeleton, further supporting CD13 as an inflammatory adhesion molecule. Mechanistically, mutation of the conserved CD13 cytoplasmic tyrosine to phenylalanine abrogated adhesion; Src, FAK, and ERK phosphorylation; and cytoskeletal alterations upon Ab cross-linking. Finally, CD13 was phosphorylated in isolated murine inflammatory peritoneal exudate cells, and adoptive transfer of monocytic cell lines engineered to express the mutant CD13 were severely impaired in their ability to migrate into the inflamed peritoneum, confirming that CD13 phosphorylation is relevant to inflammatory cell trafficking in vivo. Therefore, this study identifies CD13 as a novel, direct activator of intracellular signaling pathways in pathophysiological conditions.

Contributions of the Three CYP1 Monooxygenases to Pro-Inflammatory and Inflammation-Resolution Lipid Mediator Pathways

All three cytochrome P450 1 (CYP1) monooxygenases are believed to participate in lipid mediator biosynthesis and/or their local inactivation; however, distinct metabolic steps are unknown. We used multiple-reaction monitoring and liquid chromatography-UV coupled with tandem mass spectrometry-based lipid-mediator metabololipidomics to identify and quantify three lipid-mediator metabolomes in basal peritoneal and zymosan-stimulated inflammatory exudates, comparing Cyp1a1/1a2/1b1(⁻/⁻) C57BL/6J-background triple-knockout mice with C57BL/6J wild-type mice. Significant differences between untreated triple-knockout and wild-type mice were not found for peritoneal cell number or type or for basal CYP1 activities involving 11 identified metabolic steps. Following zymosan-initiated inflammation, 18 lipid mediators were identified, including members of the eicosanoids and specialized proresolving mediators (i.e., resolvins and protectins). Compared with wild-type mice, Cyp1 triple-knockout mice exhibited increased neutrophil recruitment in zymosan-treated peritoneal exudates. Zymosan stimulation was associated with eight statistically significantly altered metabolic steps: increased arachidonic acid-derived leukotriene B₄ (LTB₄) and decreased 5S-hydroxyeicosatetraenoic acid; decreased docosahexaenoic acid-derived neuroprotectin D1/protectin D1, 17S-hydroxydocosahexaenoic acid, and 14S-hydroxydocosahexaenoic acid; and decreased eicosapentaenoic acid-derived 18R-hydroxyeicosapentaenoic acid (HEPE), 15S-HEPE, and 12S-HEPE. In neutrophils analyzed ex vivo, elevated LTB₄ levels were shown to parallel increased neutrophil numbers, and 20-hydroxy-LTB₄ formation was found to be deficient in Cyp1 triple-knockout mice. Together, these results demonstrate novel contributions of CYP1 enzymes to the local metabolite profile of lipid mediators that regulate neutrophilic inflammation.

Putting the brakes on acute lung injury: can resolvins suppress acute lung injury?

Putting the brakes on acute lung injury: can resolvins suppress acute lung injury?

Hematopoietic Stem-Progenitor Cells Restore Immunoreactivity and Improve Survival in Late Sepsis

Sepsis progresses from an early/acute hyperinflammatory to a late/chronic hypoinflammatory phase with immunosuppression. As a result of this phenotypic switch, mortality in late sepsis from persistent primary infection or opportunistic new infection often exceeds that in acute sepsis. Emerging data support that persistence of the hypoinflammatory (hyporesponsive) effector immune cells during late sepsis might involve alterations in myeloid differentiation/maturation that generate circulating repressor macrophages that do not readily clear active infection. Here, we used a cecal ligation and puncture (CLP) murine model of prolonged sepsis to show that adoptive transfer of CD34(+) hematopoietic stem-progenitor cells after CLP improves long-term survival by 65%. CD34(+) cell transfer corrected the immunosuppression of late sepsis by (i) producing significantly higher levels of proinflammatory mediators upon ex vivo stimulation with the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide, (ii) enhancing phagocytic activity of peritoneal macrophages, and (iii) clearing bacterial peritonitis. Improved immunity by CD34(+) cell transfer decreased inflammatory peritoneal exudate of surviving late-sepsis mice. Cell tracking experiments showed that the transferred CD34(+) cells first appeared in the bone marrow and then homed to the spleen and peritoneum. Because CD34(+) cells did not affect the early-phase hyperinflammatory response, it is likely that the newly incorporated pluripotent CD34(+) cells differentiated into competent immune cells in blood and tissue, thereby reversing or replacing the hyporesponsive endotoxin-tolerant cells that occur and persist after the initiation of early sepsis.

Cell surface expression of the 300 kDa mannose-6-phosphate receptor by activated T lymphocytes.

Phosphosugars, such as mannose-6-phosphate (M6P), have been shown previously to display anti-inflammatory properties, notably inhibition of experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis in rats. It has been proposed that M6P exerts its anti-inflammatory effect by displacing lysosomal enzymes, which are involved in T-cell extravasation into inflammatory sites, from the 300 kDa mannose-6- phosphate receptor (MPR-300) on the surface of T cells. If this model is correct MPR-300 should be selectively expressed on the surface of activated T cells, as T cell entry into the central nervous system in EAE depends on the T cells being in an activated state. Thus, the present study examines whether cell surface expression of MPR-300 by T lymphocytes correlates with their state of activation and whether T cells in inflammatory sites express the receptor. Flow cytometric studies showed MPR-300 to be absent from the surface of unstimulated rat T cells isolated from peripheral blood and lymphoid tissues, and T cells resident within the peritoneal cavity. In contrast, MPR-300 was expressed on activated T cells derived from an inflammatory peritoneal exudate. In vitro studies demonstrated transient expression of MPR-300 on the surface of splenic T cells following stimulation with Con A. MPR-300 was also induced on T-cell lines by antigen stimulation. These data demonstrate that T cells in inflammatory sites express MPR-300 on their surface and activation of T lymphocytes induces cell surface expression of MPR-300. Such findings are consistent with the hypothesis that cell surface MPR-300 is required for the entry of T cells into inflammatory sites.

The membrane-bound but not the soluble form of human Fas ligand is responsible for its inflammatory activity.

The ectopic expression of Fas ligand (FasL/CD95L) in tissues or tumors induces neutrophil infiltration and the destruction of the tissues or the rejection of tumors. It has been suggested that the infiltrated neutrophils are responsible for the latter phenomena. FasL is synthesized as a type II transmembrane protein, and soluble FasL is produced by a proteolytic mechanism from the membrane-bound form. We previously demonstrated that uncleavable membrane-bound FasL of mice induces IL-1 beta release from inflammatory cells, and suggested that the IL-1 beta enhances neutrophil infiltration. However, recent papers reported that human soluble FasL is directly chemoattractive to neutrophils in vitro and proposed that the soluble form of FasL is responsible for its inflammatory activity. Therefore, in this report, we investigated which form is responsible for the inflammatory activities of human FasL. We produced tumor cell lines expressing one or both forms of human FasL. Cells expressing both forms or only the membrane-bound form of FasL induced neutrophil infiltration when transplanted into the peritoneal cavity of syngeneic mice, while cells expressing only the soluble form did not. Purified soluble FasL failed to induce neutrophil infiltration in vivo. IL-1 beta release from inflammatory peritoneal exudate and acceleration of tumor rejection were also mediated by membrane-bound but not soluble FasL. These results indicate that the membrane-bound form of FasL is primarily responsible for its inflammatory activity.

The multi-organ origin of interleukin-5 in the mouse.

Murine Ba/F3 cells were transfected with cDNA for the alpha-chain of the murine interleukin-5 (IL-5) receptor and cloned lines of these cells were able to proliferate in response to as little as 2.5 pg/ml of IL-5. The bioassay was demonstrated to be specific for IL-5 and was able to measure IL-5 produced in culture by organs from adult C57BL/6 and BALB/c mice. The highest levels of IL-5 were produced by lung tissue but thymus and bladder consistently produced IL-5 and more variable production was observed by the heart, spleen, muscle, bone shaft, uterus and testes. Bone marrow cells produced no detectable IL-5. Observed levels of production of IL-5 were similar when using organs from mice lacking high-affinity receptors for IL-5 and from nu/nu, RAG-1-/- and NOD/SCID mice lacking T lymphocytes. In inflammatory peritoneal exudates induced by the injection of casein plus bacteria, levels of induced IL-5 were higher if the mice lacked high-affinity receptors for IL-5. The data indicate that T lymphocytes are not the dominant cellular source of IL-5 in organ-conditioned media and that local IL-5 production can occur with a wide range of normal murine organs.

Mediation of the Immunomodulatory Effect of β-Estradiol on Inflammatory Responses by Inhibition of Recruitment and Activation of Inflammatory Cells and Their Gene Expression of TNF-α and IFN-γ

Background: Estrogen has long been reported to show immunomodulatory effects on immune responses, yet, its specific anti-inflammatory mechanism is not clear. Methods: In this study, we analyzed the effects of β-estradiol (E2), at its contraceptive dose, on both T cell-independent and T cell-dependent inflammations, and the associated immune mechanism, in female mice. The T cell-independent inflammation was locally induced either with an intradermal injection of olive oil in the footpad, or by an intraperitoneal injection of proteose peptone (PP). The T cell-dependent inflammation was induced by an intraperitoneal injection of the purified protein derivatives (PPD). Results: While E2 inhibited olive oil-induced inflammation as monitored by the decrease in footpad swelling, it did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by cells at the inflammatory locus. E2 also inhibited PP-induced inflammation as monitored by the decrease in the number of inflammatory peritoneal exudate cells (PEC) coinciding with a marked decrease in the number of macrophages and granulocytes (Gr. 1+). While E2 did not affect the gene expression of monocyte chemoattractant protein-1 and IL-6 by PP-elicited PEC, it decreased both gene expression and production of TNF-α. E2 also decreased the number of cells expressing the lymphocyte function-activated protein-1 in PP-elicited PEC, but not for CD62L. In purified protein derivative-induced T cell-dependent inflammation, E2 decreased the total cellularity of PEC and the relative numbers of CD3+ and CD4+ T cells, and the number of cells expressing the lymphocyte activation markers CD40, CD44, CD69 and IL-2Rα in PEC. Furthermore, while E2 did not affect the gene expression of the early T lymphocyte activation protein-1 by PEC, it decreased the gene expression of INF-γ. Conclusion: Collectively, the results suggest that E2-mediated inhibition of inflammatory responses may be due to a combination of suppression of homing and activation of inflammatory cells and their production of TNF-α and IFN-γ.

Deacylation of Lipopolysaccharide in Whole Escherichia coli during Destruction by Cellular and Extracellular Components of a Rabbit Peritoneal Inflammatory Exudate

Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals. However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established. We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS. We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting. Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone. Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria. When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred. In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h. Deacylation of LPS was always restricted to the secondary acyl chains. Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria. This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.

Beta-Chemokines enhance parasite uptake and promote nitric oxide-dependent microbiostatic activity in murine inflammatory macrophages infected with Trypanosoma cruzi.

In the present study, we describe the ability of Trypanosoma cruzi trypomastigotes to stimulate the synthesis of beta-chemokines by macrophages. In vivo infection with T. cruzi led to MIP-1alpha, RANTES, and JE/MCP1 mRNA expression by cells from peritoneal inflammatory exudate. In addition, in vitro infection with T. cruzi resulted in expression of beta-chemokine MIP-1alpha, MIP-1beta, RANTES, and JE mRNA by macrophages. The expression of the beta-chemokine MIP-1alpha, MIP-1beta, RANTES, and JE proteins by murine macrophages cultured with trypomastigote forms of T. cruzi was confirmed by immunocytochemistry. Interestingly, macrophage infection with T. cruzi also resulted in NO production, which we found to be mediated mainly by beta-chemokines. Hence, treatment with anti-beta-chemokine-specific neutralizing antibodies partially inhibited NO release by macrophages incubated with T. cruzi parasites. Further, the addition of the exogenous beta-chemokines MIP-1alpha, MIP-1beta, RANTES, and JE/MCP-1 induced an increased T. cruzi uptake, leading to enhanced NO production and control of parasite replication in a dose-dependent manner. L-NMMA, a specific inhibitor of the L-arginine-NO pathway, caused a decrease in NO production and parasite killing when added to cultures of macrophages stimulated with beta-chemokines. Among the beta-chemokines tested, JE was more potent in inhibiting parasite growth, although it was much less efficient than gamma interferon (IFN-gamma). Nevertheless, JE potentiates parasite killing by macrophages incubated with low doses of IFN-gamma. Together, these results suggest that in addition to their chemotactic activity, murine beta-chemokines may also contribute to enhancing parasite uptake and promoting control of parasite replication in macrophages and may play a role in resistance to T. cruzi infection.

Deacylation of purified lipopolysaccharides by cellular and extracellular components of a sterile rabbit peritoneal inflammatory exudate.

The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14(+) MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites.

Peroxidase activity as a measure of neutrophil populations in inflammatory peritoneal exudates of rainbow trout, Oncorhynchus mykiss (Walbaum)

A quantitative peroxidase assay using tetramethylbenzidine was applied to exudates from peritoneal cavities of rainbow trout which had been inflamed by intraperitoneal injection of Yersinia ruckeri. Most of the peroxidase activity was cell‐associated, with values for extracellular peroxidase below 5% of total peroxidase. No free peroxidase activity was detected in resting cavities. The kinetics of neutrophil numbers and peroxidase activity in peritoneal exudates during the 6 days of inflammation studied followed identical patterns, with a correlation coefficient of 0.996. This shows that the peroxidase assay can be used as an indirect, quantitative evaluation of neutrophil responses during peritoneal inflammation.

Deficiency of the Hematopoietic Cell-Specific Rho Family GTPase Rac2 Is Characterized by Abnormalities in Neutrophil Function and Host Defense

In mammals, the Rho family GTPase Rac2 is restricted in expression to hematopoietic cells, where it is coexpressed with Rac1. Rac2-deficient mice were created to define the physiological requirement for two near-identical Rac proteins in hematopoietic cells. rac2 −/− neutrophils displayed significant defects in chemotaxis, in shear-dependent L-selectin-mediated capture on the endothelial substrate Glycam-1, and in both F-actin generation and p38 and, unexpectedly, p42/p44 MAP kinase activation induced by chemoattractants. Superoxide production by rac2 −/− bone marrow neutrophils was significantly reduced compared to wild type, but it was normal in activated peritoneal exudate neutrophils. These defects were reflected in vivo by baseline neutrophilia, reduced inflammatory peritoneal exudate formation, and increased mortality when challenged with Aspergillus fumigatus. Rac2 is an essential regulator of multiple specialized neutrophil functions.

Mobilization of potent plasma bactericidal activity during systemic bacterial challenge. Role of group IIA phospholipase A2.

Extracellular mobilization of Group IIA 14-kD phospholipase A2 (PLA2) in glycogen-induced rabbit inflammatory peritoneal exudates is responsible for the potent bactericidal activity of the inflammatory fluid toward Staphylococcus aureus (1996. J. Clin. Invest. 97:250-257). Because similar levels of PLA2 are induced in plasma during systemic inflammation, we have tested whether this gives rise to plasma bactericidal activity not present in resting animals. Baboons were injected intravenously (i.v.) with a lethal dose of Escherichia coli and plasma or serum was collected before and at hourly intervals after injection. After infusion of bacteria, PLA2 levels in plasma and serum rose > 100-fold over 24 h to approximately 1 microg PLA2/ml. Serum collected at 24 h possessed potent bactericidal activity toward S. aureus, Streptococcus pyogenes, and encapsulated E. coli not exhibited by serum collected from unchallenged animals. Bactericidal activity toward S. aureus and S. pyogenes was nearly completely blocked by a monoclonal antibody to human Group IIA PLA2 and addition of purified human Group IIA PLA2 to prechallenge serum conferred potent antistaphylococcal and antistreptococcal activity equal to that of the 24 h post-challenge serum. PLA2-dependent bactericidal activity was enhanced approximately 10x by factor(s) present constitutively in serum or plasma. Bactericidal activity toward encapsulated E. coli was accompanied by extensive bacterial phospholipid degradation mediated, at least in part, by the mobilized Group IIA PLA2 but depended on the action of other bactericidal factors in the 24-h serum. These findings further demonstrate the contribution of Group IIA PLA2 to the antibacterial potency of biological fluids and suggest that mobilization of this enzyme during inflammation may play an important role in host defense against invading bacteria.

Uptake of neutrophils and neutrophilic components by macrophages in the inflamed peritoneal cavity of rainbow trout (Oncorhynchus mykiss)

The interaction between neutrophils and macrophages in peritoneal inflammatory exudates in rainbow trout injected intraperitoneally with several bacterial and non-bacterial phlogistic agents was studied by light and electronmicroscopic histochemistry. The transfer of neutrophilic components to macrophages was demonstrated by the presence in macrophages of myelo-peroxidase, an enzyme of the neutrophil not produced by rainbow trout monocytes and macrophages. Two mechanisms observed for the transfer were phagocytosis of intact neutrophils and phagocytosis of apoptotic bodies released from inflammatory neutrophils. Neutrophils with phagocytosed bacteria were also found to be ingested by macrophages, with the consequent transfer of neutrophilic components and bacteria to the macrophages. Two other mechanisms of transfer are considered. One involves the intake by the macrophage of neutrophilic granules or granular molecules released by lysing or degranulating neutrophils, respectively. The other may be through gap junctions which were observed between adhering inflammatory neutrophils and macrophages.

Neutrophil and macrophage responses to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A light and electron microscopic cytochemical study.

The neutrophil and macrophage responses that accompany inflammation were studied in the peritoneal cavity of rainbow trout Oncorhynchus mykiss using light and electron microscopic cytochemistry. Neutrophils of inflammatory peritoneal exudates were alpha-naphthyl butyrate esterase-negative, peroxidase-positive and rich in cytoplasmic glycogen granules. Macrophages were poor in glycogen, esterase-positive and usually peroxidase-negative. Some peroxidase-positive macrophages were due to the transfer to macrophages of neutrophilic peroxidase. The ultrastructural double labelling for glycogen-peroxidase or esterase/peroxidase was most useful for precisely characterising neutrophils and macrophages in the inflamed peritoneal cavities and for correctly labelling peroxidase-positive macrophages. Intraperitoneal injection of casein, Incomplete Freund's Adjuvant (IFA) and live or formol-killed Yersinia ruckeri resulted in a rapid influx of neutrophils, peaking at 24 to 48 h post-injection and reaching values, in the case of live bacteria, 500 x those in the resting, unstimulated peritoneal cavity. Peritoneal macrophages also increased, but the response was slower (peak at 5 d) and with more modest increases in number (7.5 x). Neutrophil and mononuclear cells returned to normal values after 15 d in the case of casein and bacteria, but continued above base values 30 d after the injection of IFA. Conversely, after the injection of phosphate buffered saline, India ink or with sham-injections, very moderate neutrophil and and macrophage responses subsided in a few hours. Phagocytosis of bacteria was studied by light microscopy of preparations stained for peroxidase by a new method which allows for the simultaneous observation of intracellular bacteria and peroxidase staining. When Y. ruckeri was injected into resting peritoneal cavities, bacteria were ingested by the resident macrophages. When the bacteria were injected into cavities with high numbers of neutrophils (due to the previous injection of casein), more neutrophils than macrophages contained bacteria. Results show that the macrophages are the resident phagocytes of the peritoneal cavity of trout, while neutrophils are present in that body cavity in significant numbers only in situations of inflammation and only as long as the inflammation persists.

PH-dependent regulation of leukocyte 5-lipoxygenase activity in inflammatory exudates by albumin.

In order to study the regulation of cellular 5-lipoxygenase activity under inflammatory conditions, the effects of inflammatory exudates on rat leukocyte 5 lipoxygenase activity were investigated. Peritoneal leukocytes and inflammatory exudates were collected from glycogen treated rats. Glycogen (1 g/kg body weight, in a final volume of 3 ml PBS) was injected intraperitoneally into male Wistar rats. After 4 h, the inflammatory exudate was collected. Rat peritoneal leukocytes were isolated and the cellular 5-lipoxygenase activity was determined by HPLC after cell stimulation with calcium ionophore A23187. Inflammatory exudates from glycogen treated animals strongly inhibited cellular 5-lipoxygenase activity of ionophore challenged leukocytes. Albumin was identified as the inhibitor in exudates. Inhibition of cellular 5-lipoxygenase activity by albumin was pH-dependent and was strongly increased by the alkaline pH (7.9-8.0) of the exudate. The albumin effect increased in the range of pH 7.4-8.2 where albumin undergoes a conformational change called neutral to base (N-B) transition. S-Carboxymethyl-albumin had a similar activity to that of albumin, which indicated that the free SH-group at Cys-34 of albumin is not necessary for the effect. The albumin dimer showed a significantly higher inhibition than albumin and it suppressed cellular 5-lipoxygenase activity by 98%. Peptic and tryptic fragments of albumin which comprise domains I, II and II, III, respectively, were less active or inactive. Thus, an intact albumin molecule or the dimer are required for efficient inhibition of cellular 5-lipoxygenase activity. Our data suggest that during inflammation, albumin extravasation and changes in pH-value are involved in the regulation of the inflammatory reaction by suppression of leukotriene release.

P15s (15-kD Antimicrobial Proteins) Are Stored in the Secondary Granules of Rabbit Granulocytes: Implications for Antibacterial Synergy With the Bactericidal/Permeability-Increasing Protein in Inflammatory Fluids

AbstractThe bactericidal potency toward complement-resistant Escherichia coli of bactericidal/permeability-increasing protein (BPI) released from polymorphonuclear leukocytes (PMNs) in glycogen-induced inflammatory peritoneal exudates of rabbits is dependent on synergy with extracellular p15s. This synergy depends on the high molar ratio of p15s to BPI in the extracellular fluid (∼50:1), which greatly exceeds the intracellular ratio (∼5:1). To explore the possible basis of the greater accumulation of p15s in inflammatory fluid, we examined the subcellular localization of BPI and p15 in PMNs. Immunogold electron microscopy confirmed the storage of BPI in primary granules and showed that p15s are stored in secondary granules. Reverse-transcription polymerase chain reaction of density-fractionated rabbit bone marrow cells verified that p15s are expressed later than BPI during myeloid differentiation. As the inflammatory response evolves, p15 mRNA appears earlier in blood and exudate cells than mRNA for BPI, consistent with release of progressively less mature precursors from bone marrow. Finally, Ca2+-ionophore–mediated exocytosis of p15s occurs more readily than release of BPI. We therefore propose that localization of a synergistic partner of BPI (p15s) in more readily released secondary granules allows the neutrophil to mobilize potent BPI-dependent antibacterial activity extracellularly without significant depletion of intracellular BPI stores.