Characterization of leukotriene production in vivo and in vitro in resident and elicited peritoneal macrophages in chickens and mice

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(1), 41-49

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C h a r a c t e r i z a t i o n of leukotriene production in vivo and in vitro in resident and elicited peritoneal m a c r o p h a g e s in chickens and mice J. W h e l a n , ~ K. A. G o l e m b o s k i , 2 K. S. B r o u g h t o n , ~ J. E. Kinsella, ~ R. R. Dietert 2 1Lipids Research Laboratory, Institute of Food Science, Cornell University, Ithaca, NY 2Institute for Comparative and Environmental Toxicology and Department of Poultry and Avian Sciences, Comell University, Ithaca, NY

Summary Previously, we reported differences in arachidonic acid metabolism in elicited chicken peritoneal macrophages when compared with murine resident and elicited peritoneal macrophages. 1We now describe leukotriene (LT) production in the same systems, using resident (murine) and inflammatory macrophages (from both species). Inflammatory (4- or 42-h Sephadex-elicited) peritoneal macrophages from chickens lacked the capacity to produce LT in vivo (following opsonized zymosan [OZ] stimulation) or in vitro, in response to A23187. In addition, chicken macrophages were unable to metabolize exogenously added LTC4 or LTD4 in vitro. In contrast, resident murine peritoneal macrophages produced measurable quantities of LTs (in vivo) within 5 min with an 8-fold increase after 45 min. LTC4was effectively converted to LTE4 in vivo in a time-dependent manner (65% LTC4/35% LTE4 after 5 min stimulation with OZ and 6% LTC4/94% LTE4 after 60 min stimulation), but not in vitro. The lack of LTC4 metabolism to LTE4 in vitro could not be explained by cell-cell interaction between adherent and nonadherent cells. LTD4 was not detected under any experimental condition. Mudne peritoneal cells incubated with LTD4 (with or without agonist) produced LTE4 in a time-dependent fashion. Addition of L-cysteine (a dipeptidase inhibitor) did not explain the lack of detectable levels of LTD4 following intraperitoneal stimulation with OZ. These results suggest that elicited chicken peritoneal macrophages are incapable of producing LTs compared to murine peritoneal macrophages. In addition, these studies fail to explain the different product profiles associated with in vivo stimulation of murine peritoneal macrophages as compared to in vitro stimulation.

INTRODUCTION Macrophages play an important role in modulating the inflammatory response. Following stimulation, these cells are responsible for the production and secretion of a number of proinflammatory agents, namely, reactive oxygen species, proteolytic enzymes and eicosanoids. We have characterized the functional properties of avian inflammatory macrophages and have assessed the production of inflammatory mediators, including eicosanoids derived from the cyclooxygenase pathway? The chicken has a unique system which has few, if any, harvestable Received 22 November 1995 Accepted 30 April 1996 Correspondence to: Dr Jay Whelan, Department of Nutrition, 229 Jessie Harris Building, University of Tennessee, Knoxville, TN 37996-1900,

resident peritoneal cells (PCs). Pure populations of inflammatory cells may be recruited by injection of carbohydrate irritants. When the irritant employed is crosslinked dextran (Sephadex G-50) the resulting population is primarily macrophages, with the remahnder being heterophfls (granulocytes). 2 Previously, we reported that murine peritoneal macrophages are good producers of eicosanoids (LTs and prostaglandins). 3,4 We also reported differences in arachidonic acid metabolism via the cyclooxygenase pathway in elicited chicken peritoneal macrophages when compared with murine resident and elicited peritoneal macrophages; 1 however, no information is currently available describing the ability of elicited chicken peritoneal macrophages to produce LTs. We now describe LT production in elicited chicken peritoneal macrophages as compared to murine resident and elicited macropahges (stimulated in vivo and in vitro), including 41

42

Whelan et al

the kinetics of murine LT production, focussing on relative product profiles. We also try to ascertain why routine peritoneal macrophages produce LTC4 and LTE4 in vivo, but only LTC4 in vitro with no apparent formation of LTE4. MATERIALS

AND METHODS

Materials

Sephadex G-50, calcium ionophore A23187, zymosan, rabbit serum, EDTA and PMA were purchased from Sigma Chemical (St Louis, MO). DMEM and RPMI were purchased from Gibco (Gaithersburg, MD). PGB1, LTB4,LTC4, L T D 4 and L T E 4 w e r e purchased from Cayman Chemical (Ann Arbor, MI). Animals Mice

CD-1 male mice (-30g) were used in all mice experiments. The mice were randomly divided and housed on a 12-h dark/light cycle in stainless steel cages, five animals per cage. The mice were fed Prolab mouse chow diet (Agway, RMF1000, Syracuse, NY). Food and water were provided ad libitum. All animal procedures were approved by the Comell University Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NRC 1985). Chicken

Female Cornell K strain White Leghorn chicks (4 to 5 weeks of age) were utilized in all chicken experiments. Birds were raised in wire brooder batteries; food (Cornell D ration, a modification of Cornell C)5 and water were provided ad libitum and lights were on a 15-h day schedule. Methods Preparation of opsonized zymosan

Opsonized zymosan (OZ) was prepared by the methods of Maroussem et al with the following modificationsY Briefly, zymosan at 10 mg/ml in distilled water was heated to 100°C for 1 h, then cooled and pelleted by centrifugation. The zyrnosan was then opsonized by suspension in rabbit serum at 10rag/m1 for 30min at 37°C. Following incubation, the zyrnosan was recovered by centrifugation, twice washed with saline (0.9% NaC1) and resuspended in saline at 2 mg/ml. Leukotriene analysis

LTs were isolated by solid-phase extraction using a C-18 cartridge (Burdick & Jackson, Muskegon, M0, sequentially washed with distilled water, then with hexane, and eluted off the column with methanol. Following evaporation,

the residue was reconstituted in the HPLC solvent system of methanol/water (65:35, v/v), pH 5.6, containing 5 mM ammonium acetate and 1 mM EDTA. The LTs were separated by reverse phase-HPLC on a Whatman (Hfllsboro, OR) Partisphere C-18 column (6mm x 12.5cm) with a flow rate of 0.9 ml/min. Quantification was based on molar extinction coefficients using the internal standard PGB1, with a Hewlett-Packard (Liverpool, NY) 1040A Diode Array scanning spectrophotometer, monitoring at 280 nm. All compounds were identified (detection limit of 1 ng) by their distinct UV absorption spectra and retention times were compared to known standards as reported previously with representative chromatograms. 3,4,7 LTB4 was identified by comparing retention times on RP-HPLC with that of the authentic standard and whose identity was confirmed by its characteristic UV absorption spectrum with Xm~ at 270 nm. Sephadex-elicited peritoneal cells

An inflammatory response was initiated by intraperitoneal injection of Sephadex, as previously described, s Briefly, Sephadex G-50 was weighed, then preswollen overnight in type 1 purified water. After two washes with water, the pellet was resuspended in the appropriate volume of sterile saline (0.75) to provide a 3°/0 (w/v) solution, which was injected at 1 ml/100 g body weight. Four or 42 h after injection, chickens or mice were sacrificed and peritoneal exudate cells were harvested by flushing the peritoneal cavity with 30 ml (chickens) or 6 ml (mice) cold sterile saline (0.9%) containing 0.5 U/ml heparin. Saline used for in vivo experiments also contained 1 mM EDTA and a prostaglandin B1 (PGB~) internal standard (100ng). (Resident mouse PCs were harvested in the same manner, without the prior injection of Sephadex.) Samples were centrifuged, and in some experiments the supernatants were analysed for LT formation (in vivo LT production). In other experiments, PCs were washed twice with cold 0.15 M phosphate-buffered saline (PBS; pH 7.2) prior to counting and further analysis (in vitro LT production). Cytospins were prepared and stained with May-Grunwald Giemsa for microscopic analysis of cell populations. In vivo generation and isolation of leukotrienes

In all of the following in vivo experiments, OZ (1 mg in 0.5 ml saline) was injected intraperitoneally to stimulate LT release. ~,9,~°After 30 min, the animals were sacrificed by ether inhalation (mice) or electrocution (chicken) and the peritoneal cavity was washed with saline containing 1 mM EDTA and 100 ng of PGB1 as internal standard. The peritoneal washes were centrifuged (700 x g for 4 min at 25°C) to pellet the ceils, and the supernatants were made up to a final volume of 10 ml containing 10% methanol and 1.5 mM formic acid. In those experiments where PGB~

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(1), 41-49

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Characterization of leukotriene production in elicited chicken and mice peritoneal macrophages

was not included in the initial peritoneal wash, the data was expressed as ng per mouse as described previously/ Time course for leukotriene production in vivo

Mice: A time course of LT biosynthesis by resident mouse PCs was performed by injecting OZ intraperitoneally into five otherwise untreated mice, followed by washing of the peritoneal cavity with saline at 15, 20, 30, 45 or 60 rain after OZ injection. The exudate washes were analyzed for LT production. A second time course experiment followed the same procedure except that peritoneal washes were performed at 5, 15, 30, 45 or 60 rain after OZ injection. Chickens: To examine the possibility that nonharvestable resident PCs produced measurable LT, six chickens were injected with either saline (n = 1) or OZ (n = 5) intraperitoneally. After 30 rain the chickens were sacrificed, the peritoneal cavities were flushed, and the washes were analyzed for LT production. LT biosynthesis by inflammatory (Sephadex-elicited) PCs was also examined in a time-dependent manner: 48 chicks were injected i.p. with Sephadex, then (at 4 or 42 h post-Sephadex), birds were injected i.p. with saline or OZ (3 birds per treatment at each timepoint). At 15, 30, or 60 min after the second injection, peritoneal cavities were washed for LT determination. In other experiments, fluid from peritoneal washes collected 4 or 42 h after Sephadex injection (with no further stimulus) was analyzed for LT content. Murine leukotriene production in vivo with sephadex-elicited cells

LT production in vivo by Sephadex-elicited mouse PCs, with or without OZ stimulation, was assessed. Eight mice were injected with Sephadex as described above. Four (n = 4) or 42 h (n = 4) after Sephadex injection, the mice were injected i.p. with saline (control, n = 1 per timepoint) or with OZ (n = 3 per timepoint). Thirty minutes after OZ injection, peritoneal cavities were flushed and the washes were assayed for LT production. In a separate experiment, six mice were injected with Sephadex as described above. The peritoneal cavities were flushed at 4 or 42 h after Sephadex injection, with no further stimulus administered, and LT production was assessed. In vivo inhibition of leukotriene formation with I-cysteine

(mice) L-Cysteine (0.5 ml of a 50mM solution in saline) was injected intraperitoneally 5 rain prior to injection with OZ or saline. After 30 min, the peritoneal cavity was washed and LT production was assayed as described above. Isolation of peritoneal macrophages for in vitro stimulation

In the following experiments, which were designed to assess in vitro LT production, resident macrophages © Pearson Professional Ltd 1997

43

(mice) or peritoneal exudate cells (chickens) were harvested 4 or 42 h after Sephadex injection as described above. Within each species, cells from similar time points were pooled and allowed to adhere in 6-well tissue culture plates (1.5-5 x 10~viable cells in 2 ml DMEM per well). After 1 h at 37°C (mouse cells) or 39°C (chicken cells), 5% CO2, nonadherent cells were removed by washing. The adherent cells were stimulated with 2 ml DMEM containing calcium ionophore A23187 (10g 1VI) or OZ (0.2 mg/ml) for 15 rain. Supernatants were harvested and assayed for LT formation. Separate adherence preparations (on glass coverslips) were stained with MayGrfinwald Giemsa to allow microscopic examination of cell populations present. In vitro incubation of L TC4 and L TD4 with peritoneal cells Mice: Mice resident peritoneal cells were harvested as described previously and separated by adherence as described above. Unseparated PCs, nonadherent cells, or adherent cells were incubated with LTD4 (0.25 gM) for 5 rain prior to stimulation with OZ (for 10 min or 30 min) or A23187 (30 min). Some unseparated PC samples were also incubated with 1-cysteine (10gM) (a dipeptidase inhibitor) for 5 min prior to the incubation with L T D 4. Nonadherent mouse PCs were also pooled, resuspended in 2 ml DMEM and preincubated with or without L T C 4 (0.25gM) for 5rain prior to stimulation with A23187 (10mM). Supernatants were harvested and assayed for LT formation. Chicken: Sephadex-elicited chicken PCs, 4 h (n = 3) or 42 h (n = 3), were collected and pooled as described above and macrophages were isolated by adherence. The cells were incubated for 5 rain with or without L T C 4 (0.25 gM) o r LTD 4 (0.25 gM) and then stimulated with calcium ionophore A23187 for an additional 15rain. Supernatants were harvested and assayed for LT formation.

RESULTS Leukotriene production in vivo Mice

Following OZ stimulation of PCs in vivo, LTC4, LTE 4 and their 11-trans isomers were detected in the supernatants of the peritoneal washes. Time course experiments demonstrated that initial LT production occurred within 5 rain following the introduction of OZ in vivo (Table 1). At 5 min, 65% of the total LTs formed w a s LTC4, with LTE 4 accounting for the rest. LTB4 and L T D 4 w e r e not detected in any of the supernatants. Maximum LT production was achieved by 45 rain. The contribution of LTE4 to the total LT pool increased with time of stimulation, comprising nearly all of the LTs quantitated at 60 min. No detectable levels of LT were produced when

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(1), 41-49

44

W h e l a n et al

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Characterization of leukotriene production in elicited chicken and mice peritoneal macrophages

saline was used as the peritoneal stimulant. Differential staining of the PCs revealed a cell population composed of >95% macrophages. LT production in vivo by elicited mouse PCs was also assessed following stimulation by Sephadex (i.p.) or saline injection prior to a 30 min stimulation with OZ (Table 2). Either 4 or 42 h after saline injection , the LT production profile 30 min after OZ stimulation was similar at each timepoint (4 and 42 h) to that observed in the time course experiment described above (Table 1). Again, LTB4 and LTD4 were not detected. In contrast, when mice were injected with Sephadex 4 or 42 h prior to OZ stimulation, total cysteinyl-LT production was decreased by more than 90% (Table 2). At both time points (4 and 42 h) LTE4 was the only cysteinyl-LT detected; however, 4 h after the injection of Sephadex (followed by 30 min OZ stimulation), LTB4 was also detected (approximately equivalent to that of LTE4) (Table 2). No measurable levels of LTB4 were detected in the 42 h Sephadex group stimulated with OZ.

45

Chicken

In a preliminary experiment, five chickens were injected i.p. with OZ (or saline). After 30 min the peritoneal cavities were washed and analyzed for LT production (Table 3). Upon analysis, no LT production was detected in any of the peritoneal washes. A follow-up experiment evaluated the effects of OZ stimulation of Sephadex-elicited PCs, 4 h and 42 h post Sephadex injection. The elicited cells were stimulated with OZ for 15 min, 30 min and 60 min (n = 4 for each time point) or with saline (n = 4 for each time point) (Table 3). No LTs were detected in the peritoneal washes at any time point, either with or without OZ stimulation. Leukotriene production in vitro Mice

Total (unseparated) PCs, or adherent and non-adherent fractions, from previously untreated mice were stimulated in vitro with either A23187 or OZ (Table 4). When

Table 3 A time course of intraperitoneal leukotriene (LT) production by Sephadex-elicited peritoneal cells following stimulation in vivo with opsonized zymosan (OZ) in chickens or stimulation in vitro with A23187.

Sephadex-elicted cells (h)

Peritoneal exudate fraction analyzed Agonist

Stimulation times (min)

Leukotrienes analyzed LTB4, LTC4, LTD4, LTE4

Experiment - In Vivo 0 or 4or 42

supernatant

saline or OZ (intraperitoneal)

0, 15, 30 and 60

No leukotrienes detected

E x p e r i m e n t - In Vitro 4 or 42 4 or 42

supernatant adherent cells

A23187

15 15

No leukotrienes detected No leukotrienes detected

Sample sizes for Experiment - In Vivo: n = 4 for each time point. Sample sizes for Experiment - In Vitro: n = 2-5.

Table 4 Leukotriene (LT) metabolism by murine peritoneal cells stimulated in vitro with opsonized zymosan (OZ) or A23187 in the presence or absence of L-cysteine.

Cells

n

Adherent Adherent Adherent Non-adherent Non-adherent Total PC Total PC Total PC Total PC Total PC Total PC Total PC

1 1 1 2 2 1 1 1 1 1 1 1

Total PC

1

Pre-agonist Agonist addition (t=time (t= time of addition in min) of addition)

Stimulation time LTB4 (min)

LTD4 ( t = 0) LTD4 LTC4 ( t = 0) LTD4 ( t = 0) LTD4 ( t = 0) LTD4 (t = 0) LTD4 (t = 0) L-cysteine (t = 0) LTD4 (t = 5) L-cysteine (t = 0) LTD4 ( t = 5)

OZ (t = 0) OZ ( t = 5) A23187 ( t = 5) A23187 ( t = 0) A23187 (t-- 5) A23187 OZ O Z ( t = 5) O Z (t = 5)

30 30 30 30 30 30 30 10 10 30 30

_a . -

-

30

O Z ( t = 10)

30

LTC4 LTD4 (% of total LTs)

LTE4

trace trace 10

100 90

.

.

.

100 100 trace trace trace

39 37 4 2

61 63 96 98

-

trace

72

28

-

trace

71

29

aNot detectable.

© Pearson Professional Ltd 1997

Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(1), 41-49

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Whelan et al

unseparated PC or adherent cells were stimulated with OZ, only a trace of LTC4 could be detected (Table 4). In contrast, when A23187 was the agonist, significant quantities of LTC4 were produced. No LTB4, LTD4 or LTE4 were produced in vitro following A23187 or OZ stimulation. When adherent cells were incubated with LTD4 followed by stimulation with OZ or A23187 for 30rain, all the LTD4 was converted to LTE4. Nonadherent cells were preincubated with LTC4 for 5 rain prior to A23187 stimulation to evaluate whether nonadherent cells are involved in the formation of LTD4 or LTE4 from LTC4 (Table 4). The only LT found in the cell culture medium was LTC4; no LTD4 or LTE4 formation was observed. To evaluate potential cell-cell interactions between the nonadherent and adherent fractious, total PCs were stimulated with b o t h A23187 and OZ (30 min) (Table 4). The results were identical to those observed with adherent cells. Following A23187 stimulation, LTC4 was produced with no detectable levels of LTD4 or LTE4. When OZ was used as the agonist, only trace amounts of LTC4 were detected. When LTD4 was incubated with total PCs for 10 rain in the absence of agonist, 60% of the LTD4 was converted to LTE4 and 90% was converted after 30 rain. Similarly, in the presence of OZ, 63% of LTD4 was converted to LTE4 after 10 min stimulation, and 98% after 30 rain. L-Cysteine, a potent dipeptidase inhibitor was used in some samples to inhibit the conversion of LTD4 to LTE4 (Table 4). When L-cysteine was added to total PC preparations prior to incubation with LTD4 (30 min), the conver-

sion of LTD4 to LTE4 was markedly inhibited. Less than 30% of LTD4 was converted to LTE4 in the presence or absence of OZ (compared with > 95% conversion in the absence of L-cysteine). To determine if LTD4 is formed by PCs in vivo as an intermediate to LTE4 formation, L-cysteine or saline was injected i.p. 5 rain prior to stimulation with OZ (30 rain) in vivo (Table 5). The control (saline) animal gave a typical response to OZ stimulation in vivo; however, in the presence of L-cysteine, LTC4 was the major LT formed (650/0) and no LTD4 was detected. Chicken

Four and 42 h Sephadex-elicited chicken macrophages were purified b y adherence and were stimulated in vitro with A23187 (Table 6). Both peritoneal wash supernatants and the stimulated cell supernatants were analysed for LT formation (Table 6). No detectable levels of LT were produced at either time point or in either supematant fraction. When chicken macrophages (purified from either 4 h or 42 h Sephadex-elicited PCs) were preincubated with either LTC4 or LTD4 5 min prior to A23187 stimulation (30 min), no conversion of LTC4 to LTD4, and no conversion of LTD4 to LTE4 was observed (Table 6). DISCUSSION

Sephadex-elicited peritoneal macrophages in chicken are poor producers of prostaglandins, in contrast to macrophages from other species; 1 however, LT production in

Table 5 Leukotriene (LT) production by murine peritoneal exudate cells stimulated in vivo with opsonized zymosan (OZ) in the presence or absence of L-cysteine.

Addition of Agonist inhibitor (t= time (t = time of of addition in min) addition)

Stimulation LTB 4 LTC4 time % of total LTs produced (min) (ng/10 6cells)

1

saline (t = 0)

OZ (t = 5)

30

-a

35% (235 ng/106 cells)

65% (415 ng/106 cells)

1

L-cysteine (t = 0)

OZ (t = 5)

30

-

65% (108 ng/106 cells)

35% (63 ng/106 cells)

LTD 4 LTE 4

aNot detectable.

Table 6 Leukotdene (LT) metabolism by Sephadex-elicited peritoneal exudate cells in vitro from chickens.

n n= n= n= n=

5 5 3 3

Elicited

Pre-agonist addition (t = time of addition in min)

Agonist (t = time of addition in rain)

LTB4

LTC4 LTD4 (% of total LTs)

4 42 4 42

LTC4 ( t = LTC4 ( t = LTD4 (t = LTD 4 ( t =

A23187 A23187 A23187 A23187

_b -

100 100 -

h h h h

0) 0) 0) 0)

(t= (t= (t-(t=

5) a 5) 5) 5)

LTE4

100 100

aStimulation time was 15 min. bNot detected.

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Characterization of leukotriene production in elicited chicken and mice peritoneal macrophages

these cells was not yet characterized. In contrast, resident murine peritoneal macrophages are effective producers o f LTs. 3'4'7 When resident mouse PCs were stimulated with OZ in vivo, LTC4 and LTE4 were quantitatively produced. A time course of stimulation suggested that the resident cells produced LTC4 which was subsequently converted to LTE4. The intermediate LT in this pathway, LTD4,11was not detected at any of the time points, nor was LTB4 detected. Since chickens do not contain a resident population of peritoneal macrophages, inflammatory exudate cells were elicited with Sephadex (4 and 42 h) prior to OZ stimulation in vivo. The lack of any detectable LT synthesis suggests that these cells are incapable or are poor producers of LTs as compared with murine macrophages. If LT biosynthesis occurred prior to or during the recruitment process (i.e. Sephadex elicitation), then stimulation by a second agonist (i.e. OZ) would dramatically reduce LT synthesis as lipoxygenases undergo a phenomenon known as self-catalyzed inactivationY Therefore, to establish whether Sephadex elicitation of macrophages may be responsible for the lack of LT synthesis in chickens, mice were injected i.p. with Sephadex (or saline), and 4 and 42 h elicited cells were stimulated in vivo with OZ (or saline) for 30 ntin. The 4 and 42 h saline injected (i.p.) controls produced a typical LT profile, quantitatively and qualitatively, following OZ stimulation. No LTD4 or LTB4 was detected in the peritoneal washes. In contrast, following OZ stimulation of the 4 and 42 h Sephadex-elicited cells, a >90% reduction in total cysteinyl-LT synthesis was observed compared to 4 and 42 h saline controls (stimulated with OZ). Sephadex has been demonstrated to act as a macrophage activating agent, 13 and activated macrophages release lower levels of arachidonic acid metabolites than do resident cells. 14 Due to the lack of in vivo LT production by elicited chicken cells, 4 and 42 h chicken elicited exudate cells were stimulated with A23187 in vitro. Following recovery of the cells from the peritoneal cavity no LTs were detected in any of the supernatants, suggesting that no LT formation occurred after peritoneal recruitment prior to the harvesting of the cells (or prior to in vivo stimulation with OZ). Following adherence and stimulation of the elicited cells with A23187, no LT formation was detected. When 4 and 42 h elicited cells from chickens were incubated with LTC4 or LTD4, only the LTs provided to the cell cultures were found in any of the superuatants, suggesting that these cells lack both 7-glutamyl transpeptidase and dipeptidase activities, 15and therefore lack the capacity to convert LTC4 to LTD4 and LTD4 to LTE4, respectively. Avian macrophages have been shown to produce LTs in the presence of exogenous arachidonic acid when virus transformed, ~6 but the absence of LT production during an ongoing inflammatory response as observed in the © Pearson Professional Ltd 1997

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present study suggests one of the following: (1) LT biosynthetic activity is present only at the earliest stages of monocyte-macrophage recruitment (perhaps prior to extravasation); (2) LT production occurs at very low levels as compared to murine cells; or (3) LTs are not actually produced under these conditions. A number of proteins known to mediate LT biosynthesis could be affectedY-19 Such dramatic differences in LT production among species raise interesting questions regarding the function of these substances in the regulation of an inflammatory response. 2° LTs have been credited with considerable influence in the initiation and progress of inflammation, yet the chicken appears to produce an adequate inflammatory response in the absence of detectable LTs. The dynamics of the avian inflammatory response, including the recruitment of circulating and marginated leukocytes and the progressive activation of inflammatory macrophages, are in many ways similar to those described in mammalian systems, although the relative peak times for certain macrophage functions, such as superoxide production, differ from the mouse? Disease-related inflammatory processes in chickens display a tendency toward chronicity and granuloma formation, with early formation of giant cells and perivascular lymphoid accumulation, 21-23although it is not clear how this could be related to relative absence of LTs. The absence of eosinophilia during hypersensitivity reactions in the chicken may reflect the absence of eosinophil chemotactic factor (ECF-A).24 An evolutionary advantage may have existed for the relative lack of mediators which cause smooth muscle constriction, such as LTC4, because the avian lung is structured quite differently than mammalian lung, e.g. bronchial walls lack cartilaginous support and are therefore more susceptible to collapse. 25 Resident murine PCs stimulated with A23187 or OZ in vitro produced only LTC4; no LTB4, LTD4 or LTE4 were detected. It has previously been demonstrated that adherent PCs produce only LTC4 when stimulated with A23187. 3'26'27 The adherent cell population was shown to be responsible for the LTC4 synthesis as stimulation of the nonadherent population (total cells stimulated were 16 x 106 cells) produced no detectable LT products. These results are in contrast to macrophges isolated from the rat, as rat peritoneal macrophages are able to produce LTC4, LTB4,LTD4 and LTE4.28'29 A striking feature of this murine peritoneal system is the different LT product profiles following in vivo and in vitro stimulation, namely the absence of LTE4 in the in vitro system. To ascertain the contribution of adherent and nonadherent cell interactions to LTE4 formation in vivo, nonadherent PCs were incubated with LTC4 prior to A23187 stimulation. No LTD4 or LTE4 formation was observed. These data suggest that adherent and nonadherent cells do not contain a functional ~-glutamyl

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transpeptidase. W h e n a d h e r e n t or total PCs were incub a t e d w i t h LTD4 for 30 m i n (with or w i t h o u t A23187 or OZ stimulation) v i r t u a l l y all of t h e LTD4 was c o n v e r t e d to LTE4. This was a t i m e - d e p e n d e n t process; t h a t is, t h e conv e r s i o n of LTD4 to LTE4 was limited w h e n t h e i n c u b a t i o n time was r e d u c e d to 10 min. I n h i b i t i o n of this c o n v e r s i o n b y t-cysteine i n d i c a t e d t h a t t h e s e cells c o n t a i n e d signific a n t quantities of dipeptidase, t h e e n z y m e r e s p o n s i b l e for t h e p r o t e o l y t i c cleavage of t h e glycyl r e s i d u e from LTD4 .as In an effort to establish t h a t LTC4 is c o n v e r t e d to LTE4 via LTD 4 in vivo, r e s i d e n t m u f i n e p e r i t o n e a l cells were stimul a t e d w i t h OZ (30 mill) in vivo in t h e p r e s e n c e or a b s e n c e of L-cysteine. Since L-cysteine e n h a n c e s LTC4 c o n v e r s i o n to LTD4 a n d inhibits LTD4 c o n v e r s i o n to LTE4, i n c u b a t i o n w i t h this i n h i b i t o r prior to OZ s t i m u l a t i o n s h o u l d e n h a n c e LTD4 formation. 28 The O Z - s t i m u l a t e d control a n i m a l p r o d u c e d a typical LT p r o d u c t profile; 3,4 however, t h e i.p. injection of L-cysteine prior to OZ s t i m u l a t i o n r e s u l t e d in a n i n h i b i t i o n of t h e c o n v e r s i o n of LTC4 (65%) to LTE4 (35%), b u t n o d e t e c t a b l e quantities of LTD4 were present. In s u m m a r y , we h a v e p r e v i o u s l y d e m o n s t r a t e d t h a t S e p h a d e x - e l i c i t e d p e r i t o n e a l cells from t h e c h i c k e n are c a p a b l e of p r o d u c i n g e i c o s a n o i d s via t h e c y c l o o x y g e n a s e p a t h w a y . However, c o n t r a r y to w h a t h a s b e e n o b s e r v e d in m u r i n e p e r i t o n e a l cells, Sephadex-elicited p e r i t o n e a l cells from t h e c h i c k e n do n o t p r o d u c e a p p r e c i a b l e q u a n tities of LTs u p o n stimulation, n a m e l y LTB4, LTC4, LTD4 or LTE4, a n d are i n c a p a b l e of c o n v e r t i n g LTC4 to LTD4 a n d LTD4 to LTE4. However, this i n a b i l i t y to p r o d u c e LTs does n o t a p p e a r to p r e v e n t t h e c h i c k e n from p r o d u c i n g a n a d e q u a t e i n f l a m m a t o r y response. M u r i n e p e r i t o n e a l cells, o n t h e o t h e r h a n d , were relatively g o o d p r o d u c e r s of LTs, b u t t h e p r o d u c t profiles are d e p e n d e n t u p o n t h e c i r c u m s t a n c e s u n d e r w h i c h t h e cells were stimulated. For example, cells s t i m u l a t e d in vitro o n l y p r o d u c e d LTC products, while in vivo s t i m u l a t i o n r e s u l t e d in t h e f o r m a t i o n of LTC a n d LTE p r o d u c t s (where LTE b e c a m e t h e d o m i n a n t LT formed). F u r t h e r more, LTD p r o d u c t s were n e v e r detected. The m u r i n e p e r i t o n e a l cells were i n c a p a b l e of c o n v e r t i n g LTC4 to LTD 4 (suggesting a lack of 7-glutamyl transpeptidase), b u t effectively c o n v e r t e d LTD4 to LTE4 in a t i m e - d e p e n d e n t manner.

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