Inflammatory Peritoneal Exudate Research Articles

The potent anti-Staphylococcus aureus activity of a sterile rabbit inflammatory fluid is due to a 14-kD phospholipase A2.

The cell-free fluid (ascitic fluid, AF) of a sterile inflammatory peritoneal exudate elicited in rabbits is potently bactericidal for complement-resistant gram-negative as well as gram-positive bacterial species. This activity is absent in plasma. We now show that essentially all activity in AF against Staphylococcus aureus is attributable to a group II 14-kD phospholipase A2 (PLA2), previously purified from AF in this laboratory. Antistaphylococcal activity of purified PLA2 and of whole AF containing a corresponding amount of PLA2 was comparable and blocked by anti-AF-PLA2 serum. At concentrations present in AF (approximately 10 nM), AF PLA2 kills > 2 logs of 10(6) S. aureus/ml, including methicillin-resistant clinical isolates, and other species of gram-positive bacteria. Human group II PLA2 displays similar bactericidal activity toward S. aureus (LD90 approximately 1-5 nM), whereas 14-kD PLA2 from pig pancreas and snake venom are inactive even at micromolar doses. Bacterial killing by PLA2 requires Ca2+ and catalytic activity and is accompanied by bacterial phospholipolysis and disruption of the bacterial cell membrane and cell wall. These findings reveal that group II extracellular PLA2, the function of which at inflammatory sites has been unclear, is an extraordinarily potent endogenous antibiotic against S. aureus and other gram-positive bacteria.

Induction of apoptotic cell death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells.

We have previously shown that the calcium-binding protein complex, calprotectin, purified from rat inflammatory peritoneal cells exerts marked cytotoxic activity against rat, mouse, and human tumor cells. We studied here whether the cytotoxicity is caused by induction of apoptosis, using mouse EL-4 lymphoma and human MOLT-4 leukemia lines as targets. The rat calprotectin sample inhibited [3H]thymidine incorporation into these cells by partially 24 h and almost completely in 48 h of culture at concentrations of 100-200 micrograms/ml. Morphological changes, that is, loss of cell volume and nuclear condensation and/or fragmentation, appeared in both cell types cultured with calprotectin from 20 h, and such apoptotic cells subsequently increased in number to compose the great majority of the cells at 40 h. Cell death, measured by stainability with trypan blue, lagged behind the emergence of the apoptotic morphology by about 2 and 10 h in EL-4 and MOLT-4 cells, respectively. DNA fragmentation was observed in EL-4 cells cultured with calprotectin, whereas it was not observed in MOLT-4 cells, consistent with results of flow cytometry showing that loss of cell DNA content caused by the factor was greater in EL-4 cells. The data indicate that calprotectin induces the apoptosis of certain tumor cells but that the occurrence of DNA fragmentation is dependent on cell type. Finally, the apoptosis-inducing activity of the calprotectin sample was abrogated by the presence of 10 microM zinc, whereas it was not affected by 5 mM calcium or magnesium.

Purification and characterization of the cytotoxic factor in rat peritoneal exudate cells: its identification as the calcium binding protein complex, calprotectin

We previously reported the existence of a growth inhibitory factor for mitogen-stimulated lymphocytes and murine tumor cell lines, MM46 and L-929, in inflammatory polymorphonuclear leukocytes. In this study, by using mouse MM46 mammary carcinoma as target, we purified the inhibitor from lysate of rat inflammatory peritoneal exudate cells by ammonium sulfate precipitation, gel filtration, isoelectrofocusing, and anion exchange chromatography. Although the in vitro inhibitory activity for MM46 growth was partitioned into three peaks in the final step, it was found that these inhibitory samples all consist of 8- and 13-kDa peptides. Analysis of amino acid sequences revealed that the partial sequences of the 8- and 13-kDa peptides completely agree with the smaller and larger components of rat calprotectin, which are predicted from cDNA, respectively, suggesting the cell growth inhibitory factor is calprotectin. In addition to MM46, the partially purified calprotectin inhibited the growth of a rat, three mice, and a human tumor cell line in similar dose-response relationships in vitro. Moreover, it exerted a cytolytic effect against all examined tumor cells. It was confirmed that the purified calprotectin induces growth inhibition and the lysis of MM46 cells and that the minimum effective concentration is between 50 and 100 micrograms/ml. The factor also inhibited the growth of bone marrow cells and macrophages. These results suggest that calprotectin is a negative regulatory factor for the growth and/or survival states of normal and tumor cells.

Differential regulation of elicited-peritoneal macrophage 14 kDa and 85 kDa phospholipase A 2(s) by transforming growth factor-β

Elicited guinea pig macrophages collected from inflammatory peritoneal exudate release soluble 14 kDa phospholipase A 2 (PLA 2) and prostaglandin E 2 (PGE 2) into surrounding media during culture (Marshall et al. (1994) J. Lipid Med. 10, 295–313). The effect of transformation growth factor β 1 (TGFβ), an immunoregulatory growth factor, was examined in this system. Exposure of cultured macrophages to TGFβ reduced both the activity and protein levels of 14 kDa PLA 2 measured in conditioned media. This inhibition occurred within the first 6–8 h, was prevalent through 72 h of exposure and was dependent on TGFβ concentration. The reduction, however, never reached more than 40–60%. Evaluation of the cellular PLA 2 activity confirmed the existence of an immunologically-related 14 kDa PLA 2 (ELISA) in the particulate fraction and an 85 kDa PLA 2 (Western analysis) in the cytosol. Exposure to TGFβ halved the particulate activity and protein levels of 14 kDa PLA 2 which was consistent with the reduction in the secreted form. Alternatively, TGFβ induced an increase in cytosolic 85 kDa PLA 2 (activity and protein) which was not apparent until 12 h and significant at 20–24 h of exposure. This demonstrates that TGFβ differentially regulates the production of these two enzymes. Despite this, neither PGE 2 synthesis nor the up-regulated cyclooxygenase −II were altered by TGFβ treatment suggesting that maximal prostanoid synthesis had been reached.

Extracellular accumulation of potently microbicidal bactericidal/permeability-increasing protein and p15s in an evolving sterile rabbit peritoneal inflammatory exudate.

To what extent the host defense role of granule-associated antibacterial proteins and peptides of PMN includes extracellular action has not been established. To address this question, we have analyzed the antibacterial activity of cell-free (ascitic) fluid (AF) obtained from glycogen-induced sterile inflammatory rabbit peritoneal exudates in which > 95% of the accumulating cells are PMN. AF, but not plasma collected in parallel, exhibits potent activity toward serum-resistant Gram-negative and Gram-positive bacteria. Total and specific antibacterial activity of AF increases during the first 12 h after injection of glycogen in parallel with the influx of PMN. At maximum, > 99% of 10(7) encapsulated Escherichia coli and Staphylococcus aureus are killed in 30 min/ml of AF. Neutralizing antibodies against the bactericidal/permeability-increasing protein (BPI) of PMN abolishes activity of AF toward encapsulated E. coli but has no effect on activity vs staphylococci. However, BPI alone (approximately 1 microgram/ml in AF) can only account for < or = 20% of AF activity toward E. coli. AF also contains 15 kD PMN proteins (p15s) that act in synergy with BPI. Purified BPI and p15s, in amounts present in AF, reconstitute the growth-inhibitory activity of AF toward encapsulated E. coli. These findings show for the first time an extracellular function of endogenous BPI, providing, together with the p15s, a potent microbicidal system toward Gram-negative bacteria resistant to plasma-derived proteins and phagocytes in inflammatory exudates.

An elicited intraperitoneal inflammatory response has no effect on the establishment of pregnancy in the mouse

To determine the impact of intraperitoneal inflammation on reproduction in the mouse. The effect of an elicited sterile intraperitoneal inflammatory exudate and the passive intraperitoneal transfer of activated syngeneic leukocytes on mating efficiency and uterine implantations was evaluated in mice. Research laboratory. Intraperitoneal injection of thioglycolate was used to elicit large numbers of activated peritoneal macrophages (mean 24.4 x 10(6) leukocytes/animal) in female CD-1 mice. The impact of this intraperitoneal exudate on mating efficiency and number of uterine horn implantations after gonadotropin-stimulated ovulation was determined. In separate experiments, the ovarian bursa present in this species was opened surgically to provide direct access of peritoneal constituents to the genital tract and the experiments repeated. Identical endpoints were evaluated in a third group of experiments using C3H/HEN syngeneic mice after passive transfer of 2, 5, and 10 x 10(6) similarly activated syngeneic peritoneal leukocytes. Neither the elicitation of a peritoneal inflammatory exudate nor the passive transfer of up to 10 x 10(6) activated syngeneic peritoneal macrophages reduced the mating efficiency or the number of uterine implantations. Furthermore, surgically opening the ovarian bursa did not alter these results, although it was associated with anatomic distortion and lowered the number of implantations in all groups. We could not confirm the previously published reports suggesting a profound adverse impact of intraperitoneal inflammation on reproduction in mice, even when providing direct continuity between the peritoneal cavity and the genital tract. Consequently, the usefulness of this model needs to be re-evaluated before considering it an adequate paradigm for evaluating potential mechanisms of infertility in women.

Purification and assay of bactericidal/permeability-increasing protein

This chapter discusses the purification and assay of bactericidal/permeability-increasing protein (BPI). BPI has been detected only in PMNs and PMN precursors and has been isolated from human, rabbit, and cow PMNs. The BPI content of these cells is ca. 60 μg/108 PMNs. Thus, greater than milligram quantities of BPI can be readily obtained from suitable starting material. Approximately 109 PMNs/animal can be collected in nearly homogeneous form from acute inflammatory peritoneal exudates of rabbits 6 to 15 hr after intraperitoneal injection of 250 to 300 ml of sterile physiological saline supplemented with glycogen (ca. 1–2 mg/ml). PMNs can also be isolated from peripheral blood. BPI can be extracted from isolated granules or from whole cell lysates. Granule-rich fractions can be prepared by disruption of cells at 0° to 4° under osmotically protective conditions and differential centrifugation. Two different methods of cell lysis have been used: (1) Homogenization of PMNs in 0.34 M sucrose using a Potter–Elvehjem homogenizer with glass mortar and motor-driven Teflon-coated pestle; (2) N2 cavitation of PMNs equilibrated in a high-pressure bomb at 400 psi in relaxation buffer minus ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid [EGTA: 100 mM KCl, 3 mM NaC1, 1 mM ATP(Na)2,3.5 mM MgCl2, 10 mM PIPES, pH 7.3].

Characterization of cell growth-inhibitory factor in inflammatory peritoneal exudate cells of rats.

We characterized the nature and reaction mode of the cell growth-inhibitory factor (here designated CGIF) from rat peritoneal exudate cells (PEC). The soluble fraction separated from the lysate of Enterococcus faecalis-induced 24 hr PEC completely inhibited Con A-induced thymocyte mitogenesis. Gel filtration chromatography showed that CGIF has a molecular weight of approximately 23-25 kDa. Isoelectric focusing with Rotofor indicates that the factor has an isoelectronic point of 5.8-6.4. CGIF was inactivated by treatment at 70 C, for 30 min or by tryptic digestion, but the activity was not destroyed by the reduction with dithiothreitol. As well as thymocyte proliferation, CGIF completely suppressed 3H-thymidine incorporation of splenocytes which were stimulated by either Con A or LPS, suggesting the factor is effective on both T and B cells. The acting point of the inhibitor appeared to be a later stage of the lymphocyte activation sequence, since it was still effective when added 28.5 hr after the addition of Con A. CGIF also reduced the viability of these cells when added with mitogens such as Con A or LPS. CGIF thus appears to be distinct from interleukin-1 receptor antagonist or transforming growth factor-beta.

Mouse MRP8 and MRP14, Two Intracellular Calcium-Binding Proteins Associated With the Development of the Myeloid Lineage

AbstractMRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.

Mouse MRP8 and MRP14, two intracellular calcium-binding proteins associated with the development of the myeloid lineage

MRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.

Comparison of the peritoneal cells elicited by oxidized regenerated cellulose (Interceed) and expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane) in a murine model

The peritoneal fluid cell responses to the available barriers used to prevent postoperative adhesions, oxidized regenerated cellulose (interceed) and expanded polytetrafluoroethylene (Gore-Tex Surgical Membrane), were compared. Oxidized regenerated cellulose and expanded polytetrafluoroethylene were inserted into the peritoneal cavity of mice and the peritoneal fluid cell number, differential cell count, morphologic type, adherence to plastic, and phorbol-12,13-myristate acetate-stimulated hydrogen peroxide production evaluated. Peritoneal fluid cell attachment to oxidized regenerated cellulose and expanded polytetrafluoroethylene was evaluated by electron microscopy. Oxidized regenerated cellulose promptly elicited increased numbers of large, morphologically activated peritoneal fluid macrophages that adhered more rapidly and produced more hydrogen peroxide than controls. Expanded polytetrafluoroethylene gradually increased the number of normal-appearing peritoneal fluid macrophages with increased hydrogen peroxide production but normal adherence. Macrophages rapidly attached to both materials in vivo, with oxidized regenerated cellulose being rapidly degraded. Oxidized regenerated cellulose, to a greater extent than expanded polytetrafluoroethylene, elicits an acute peritoneal fluid inflammatory exudate in the mouse, which may contribute to the oxidized regenerated cellulose-induced peritoneal injury and de novo adhesions observed in this model.

Isolation of rat peritoneal mononuclear and polymorphonuclear leucocytes on discontinuous gradients of Nycodenz

Centrifugation of rat leucocytes from thioglycollate-induced inflammatory peritoneal exudate on a discontinuous gradient of Nycodenz with a density of 1106 g/l and an osmolarity of 400 mosmol/1 separated the polymorphonuclear from the mononuclear leucocytes. The cells on the interphase between the buffer and the gradient medium contained 96% mononuclear leucocytes with a recovery of >60%, and the bottom fraction consisted of 98% polymorphonuclear leucocytes with a yield of 91%, when an exudate isolated 20 h after the injection of thioglycollate was fractionated. Leucocytes isolated from non-inflamed rat peritoneum could be enriched in the fraction of nonspecific esterase-positive cells from 86% to 96% with a recovery of 82% on a gradient with a density of 1091 g/1 and an osmolarity of 325 mosmol/1. The viability of the isolated cells was > 95% (trypan blue exclusion test),and there was no measurable reduction in the fraction of phagocytosing cells (latex and opsonized zymosan) after exposure to the hypertonic gradient material.

Exacerbation of murine listeriosis by a monoclonal antibody specific for the type 3 complement receptor of myelomonocytic cells. Absence of monocytes at infective foci allows Listeria to multiply in nonphagocytic cells.

Treatment of mice with a rat mAb (5C6) specific for an epitope of the type 3 complement receptor of myelomonocytic cells severely interfered with the ability of the mice to resist infection with Listeria monocytogenes. Consequently, a sublethal infection was rapidly converted to a lethal one that resulted in death in 5 d. However, infection was only exacerbated if 5C6 was given earlier in infection, before mononuclear phagocytes populated sites of Listeria implantation in the liver and spleen. If given after day 3 of infection, 5C6 caused only a temporary increase in bacterial multiplication. The infection-enhancing effect of 5C6 was associated with failure of mice to focus mononuclear phagocytes at sites of bacterial multiplication of Listeria in liver hepatocytes and extracellulary in the spleen. This resulted in unrestricted multiplication of Listeria in hepatocytes and extracellularly in the spleen. The results are in keeping with the ability of 5C6 to inhibit the accumulation of myelomonocytic cells in peritoneal inflammatory exudates, as revealed by a previous study.

Purification and partial sequence of rabbit polymorphonuclear leukocyte-derived lymphocyte proliferation potentiating factor resembling IL-1 beta.

A rabbit polymorphonuclear leukocytes (PMN)-derived lymphocyte proliferation-potentiating factor (PMN factor) was finally purified to homogeneity. PMN factor was released from early inflammatory peritoneal exudate cells (98% of PMN) stimulated with kaolin under roller bottle culture conditions. PMN factor was purified by large sequential scale steps, using membrane-type ion exchangers and gel filtration, followed in this order by HPLC steps with cationic ion exchangers and a hydroxylapatite column. Homogeneity was manifested based on the criteria of a single m.w. 18, 500 band on silver-stained polyacrylamide gel, a superimposable activity on a UV absorbance peak in analytic HPLC gel filtration, and detection of a single amino-terminal sequence. The homogeneous PMN factor had an isoelectric value of 7.2 and an activity of 1.9 x 10(7) U/mg in the thymocyte comitogenic assay. PMN factor stimulated one-half of the maximal response of thymocyte proliferation at 2.8 x 10(-12) M. Because of similarities in the physicochemical properties, specific activity, and amino-terminal sequence between rabbit PMN factor and human IL-1 beta, this PMN factor is therefore considered to be a rabbit IL-1 beta.

Stimulated release of neutral proteinases elastase and cathepsin G from inflammatory rat polymorphonuclear leukocytes.

Rat leukocytes from inflammatory peritoneal exudates respond in vitro to a variety of chemotactic and phagocytic stimuli by releasing both elastase and cathepsin G neutral proteinase enzyme activities. PAF, FMLP, and PMA stimulated a rapid, cytochalasin B-dependent, dose-related release of both enzymes; however, leukotriene B4 was inactive. It was not possible to measure the activity of zymosan-activated serum on these cells as rat serum contains high levels of proteinase inhibitors. The calcium ionophore A23187 stimulated a dose-related, time-dependent, cytochalasin B-independent enzyme release. Concanavalin A stimulated a weak, nondose-related release of enzyme activity. Zymosan and serum-coated zymosan stimulated enzyme secretion which was markedly inhibited by the presence of cytochalasin B. These data indicate that release of azurophillic granule neutral proteinases from rat inflammatory leukocytes can be detected and quantitated in vitro. This model could provide a test system for monitoring the pattern and specificity of enzyme release from azurophil granules. The ability of a variety of stimuli to induce proteolytic enzyme release from inflammatory neutrophils may be of considerable relevance to chronic inflammatory diseases.

Structural and functional properties of a phospholipase A2 purified from an inflammatory exudate

The cell-free supernatant of sterile inflammatory peritoneal exudates contains a phospholipase A2 that participates in the digestion of Escherichia coli killed by polymorphonuclear leukocytes or by the purified bactericidal/permeability increasing protein (BPI) of these cells. This phospholipase A2 has been purified, and the sequence of the NH2-terminal 39 amino acids has been determined and compared with sequences of both BPI-responsive and BPI-nonresponsive phospholipases A2 from snake venoms and mammalian pancreas. The high concentration and location of basic residues in the NH2-terminal region is a common feature of BPI-responsive phospholipases A2 and may characterize those phospholipases A2 participating in inflammatory events.

Leukotriene B 4 and 20-OH-LTB 4 in purulent peritoneal exudates demonstrated by GC-MS

Inflammatory peritoneal exudates from 11 patients with purulent peritonitis or non-perforative appendicitis were analyzed by gas chromatography mass spectrometry (GC-MS) for their content of 5S, 12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB 4) and its ω-oxidized catabolite, 5S,12R,20-triliydroxy-6,8,10,14-eicosatetraenoic acid (20-OH-LTB4). Eleven samples of peritoneal exudates were examined. LTB 4 and 20-OH-LTB 4 were demonstrated only in the samples from patients with purulent peritonitis. LTB4 was detected in 4 samples, and 20-OH-LTB 4 was detected in 6 samples by GC-MS with selected ion monitoring. In addition, a nearly full spectrum corresponding to that of synthetic 20-OH-LTB 4 was obtained with 3 samples. Neither LTB 4 nor 20-OH-LTB 4 were detected in samples from patients with non-perforative appendicitis. LTB 4 or 20-OH-LTB4 may be related to the pathophysiological mechanism of purulent inflammations.

Kinetics of macrophage recruitment and turnover in peritoneal inflammatory exudates induced by Salmonella or thioglycollate broth.

Kinetics of peritoneal macrophage turnover during infection of mice with Salmonella enteritidis or following injection with thioglycollate broth or other peritoneal stimulants has been studied. Single intravenous injections of tritiated thymidine were given and the cells were examined by autoradiography. Maximum labelling of small adherent peritoneal macrophages occurred when 3H-thymidine was given 1 d after Salmonella and the cells were harvested 1 d later. Labelled cells decreased at later times despite maintenance of high numbers of macrophages in the exudates. Results from experiments in which labelled peritoneal cells were reinjected indicated that small, monocyte-enriched, labelled cells were not the major source of the large macrophages. Similar labelling at 2 d was observed using heat-killed Corynebacterium parvum or lipopolysaccharide (LPS) as ip stimulants. Following injection of thioglycollate broth, labelled peritoneal macrophages were only detectable if 3H-thymidine was given before the stimulant. These labelled cells remained longer in the peritoneal cavity. Labelling of and numbers of blood monocytes were consistent with the promotion of monocytopoiesis by Salmonella but not by thioglycollate. The response to thioglycollate but not Salmonella was dependent on the age of the mice. Animals injected with thioglycollate 1 d before Salmonella also had decreased resistance to bacteria and low numbers of labelled peritoneal macrophages. We propose that thioglycollate may recruit from a subset of preformed monocytes and temporarily block monocytopoiesis or macrophage bactericidal activity.

The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF 1

The feline c-fms proto-oncogene product is a 170 kd glycoprotein with associated tyrosine kinase activity. This glycoprotein was expressed on mature cat macrophages from peritoneal inflammatory exudates and spleen. Similarly, the receptor for the murine colony-stimulating factor, CSF-1, is restricted to cells of the mononuclear phagocytic lineage and is a 165 kd glycoprotein with an associated tyrosine kinase. Rabbit antisera to a recombinant v-fms-coded polypeptide precipitated the feline c-fms product and specifically cross-reacted with a 165 kd glycoprotein from mouse macrophages. This putative product of the murine c-fms gene exhibited an associated tyrosine kinase activity in immune complexes, specifically bound murine CSF-1, and, in the presence of the growth factor, was phosphorylated on tyrosine in membrane preparations. The murine c-fms proto-oncogene product and the CSF-1 receptor are therefore related, and possibly identical, molecules.

Inhibition of plasmacytoma development in BALB/c mice by indomethacin.

Indomethacin given continuously in the drinking water (20 micrograms/ml) to BALB/cAn pi mice during the latent period of pristane-induced plasmacytoma development dramatically reduced the plasmacytoma incidence from 34.9 to 2.2%. Additionally, indomethacin given from day 0 to 120 or begun as late as 60 d after a single injection of 1.0 ml pristane was also highly effective in reducing the development of plasmacytomas. Indomethacin treatment did not prevent the formation of a peritoneal inflammatory exudate or peritoneal oil granulomatous tissue, although it had a mild inhibitory effect on the intensity of the cellular inflammation, particularly after extensive treatment of greater than 100 d. Indomethacin treatment reduced the incidence of arthritis by 50%. A major effect of indomethacin treatment was a reduction in the appearance of microscopic plasmacytomas that appear in the oil granuloma before plasmacytomas can be detected by routine sampling of the peritoneal exudate. Between days 116 and 181, 16 of 20 mice given 0.5 ml pristane were found to have foci of plasmacytoma cells, while only 2 of 20 indomethacin-treated mice had foci-containing plasmacytoma cells. The number of mice with microscopic foci in the pristane-treated group greatly exceeded the expected incidence of plasmacytomas (22%) at this dose of pristane. The growth of primary plasmacytomas in transplant that is dependent on the pristane-conditioned peritoneal environment was not inhibited by indomethacin treatment. The role of indomethacin in inhibiting plasmacytoma development was not established; two possibilities are that it inhibits production of mutagenic and tissue destructive oxidants by inflammatory cells, and it inhibits prostaglandin synthesis and intracellular production of oxidant biproducts.