Determine the Vascular Permeability Induced ... - Europe PMC

Complement and Polymorphonuclear Determine the Vascular Permeability

Leukocytes Induced

Do

Not

Intraocular LPS

EDWARD L. HOWES, Jr., MD, KEYE L. WONG, MD, KAIJA T. HARTIALA, MD, ROBERT 0. WEBSTER, PhD, and JAMES T. ROSENBAUM, MD

From the Departments of Pathology and Medicine, University of California, San Francisco, California; the Department of Medicine, St. Louis University School of Medicine, St. Louis, Missouri; and the Kuzell Institute for Arthritis Research, Medical Research Institute of San Francisco, San Francisco, California

The intravitreous injection of an endotoxin of Escherichia coli 055:B5 (LPS; 0.1-0.5 pig/50 ,l. of saline) induces ocular inflammation in rabbits that is maximal 20-24 hours later and disappears by 4 days. The inflammation is characterized by an alteration in ocular vascular permeability (OVP) measured by the ocular extravasation of '25I-albumin and an outpouring of leukocytes, most of which are polymorphonuclear leukocytes (PMNs), as determined by histopathologic study. Nitrogen mustard (mechlorethamine, 1.75 mg/kg) administered 3 days prior to LPS virtually eliminates PMNs in the circulation and those infiltrating ocular tissues 20 hours after intravitreous LPS, and yet the average increase in vascular permeability is not different from that of controls. Cobra venom factor (CVF; 300-400 units) 7 hours before intravitreous LPS produces a greater than 90% decrease in both hemo-

lytic complement activity and zymosan-inducible serum chemotactic activity; yet 20 hours after LPS, the OVP is the same in CVF-treated rabbits and controls. For comparison, an ocular passive Arthus reaction (ovalbumin-anti-ovalbumin) was significantly affected by CVF pretreatment. Chemotactic activity in the aqueous humor is found in both CVF-treated and control rabbits 20 hours after intravitreous LPS. This activity attracts rabbit, but not human, PMNs, is partially heat-sensitive, and is not inhibited when PMNs are preincubated with C5a. These results indicate that neither PMNs nor circulating complement determine the OVP following intravitreous LPS, and that the chemotactic activity present in aqueous humor at the height of the inflammatory response is not primarily C5a. (Am J Pathol 1985, 118:35-42)

BACTERIAL lipopolysaccharide (LPS) injected into the vitreous of animal eyes produces a marked inflammatory response characterized by an alteration in vascular permeability and an outpouring of inflammatory cells, primarily polymorphonuclear leukocytes (PMNs). 1-3 In several examples of experimental inflammation the presence of PMNs greatly enhances the alteration in vascular permeability,4 and their removal sharply curtails the response.4-6 Ocular tissues are sufficiently sensitive to LPS that minute quantities (nanograms) can produce a response.7 This capacity for LPS to induce inflammation in the eye is commonly employed in ophthalmic research,3'7'8 but the exact mechanisms involved are not known. It is often assumed that complement activation is most important,3 but LPS activates most recognized mediators and formed elements involved in the inflammatory process.9 The complement system is certainly activated,9

but many responses due to circulating LPS such as the generalized Shwartzman reaction0'1' and endotoxin shock'2"13 need not depend on this activation. We undertook the following study to determine whether the presence of PMNs does in fact contribute to the ocular vascular permeability observed following the intravitreous injection of LPS and whether comple-

Supported in part by USPHS Grant EY-00056-13. James T. Rosenbaum is the recipient of National Institutes of Health New Investigator Research Award, AM 33950. Dr. Hartiala was supported in part by the Emil Aaltonen Foundation and the Yrjo Jahnsson Foundation. Accepted for publication July 25, 1984. Address reprint requests to Edward L. Howes, Jr., MD, Department of Pathology, San Francisco General Hospital, San Francisco, CA 94110.

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HOWES ET AL

ment activation is important in this response. It was found that neither PMNs nor circulating complement is necessary for the reaction to occur. Furthermore, the major portion of the chemotactic activity which can be identified in the aqueous fluid during the maximal inflammatory response clearly differs from C5a.

Materials and Methods Animals and Injection Schedules Outbred New Zealand white rabbits of both sexes weighing 1.8-2.2 kg were used throughout these studies. A Boivin extract of Escherichia coli 055:B5 (Difco) was injected in the vitreous of the right eye (0.1-0.5 ,Mg of LPS in 50 MI of sterile saline), while 50 MI of sterile saline was injected in the left eye. Care was taken to avoid the lens and place the injection in the center of the vitreous. This LPS in these quantities produced an inflammation by slit lamp biomicroscopy which was maximal at 24 hours following injection, and which was gone by 3-4 days. Measurement of ocular vascular permeability (OVP) was made over a 2-hour period at 20 hours after LPS injection. For this purpose 10-15 pCi of 1251_ albumin (125I-ALB, 1-2 mg; Mallinckrodt, St. Louis, Mo) was injected; 2 hours thereafter heart blood was obtained in a heparinized syringe; and the rabbits were killed by pentobarbital overdose. The eyes were enucleated under standardized conditions. 14 Radioactivity was determined for cardiac plasma and for the intact whole eyes. The enucleated eyes were carefully trimmed of excess tissues. Ocular vascular permeability was expressed as an ocular albumin space (OAS)"5:

OAS-2 hours _ CPM125I-ALB/g eye tissue (jI/g eye tissue) CPM125I-ALB/lAl cardiac plasma In previous studies, we demonstrated that the ocular albumin space reflects primarily extravascular albumin and that the calculated blood volume of the rabbit eye is responsible for less than 250o of the total radioactivity. 16 Many of these same eyes were fixed in 4% formaldehyde-iWo glutaraldehyde in a phosphate buffer (pH 7.2); portions embedded in glycol methacrylate, sectioned on a Sorvall JB-4 microtome, and stained with hematoxylin and eosin, methylene blue and azure II, and Giemsa.

Passive Arthus Reactions In order to evaluate further the ocular effects of complement depletion (see below), an ocular passive Arthus reaction was established and assessed in the manner just described. For this purpose an ammonium sulfate fraction of pooled hyperimmune rabbit anti-

AJP * January 1985

ovalbumin antiserum was injected intravenously in normal rabbits (1.5 mg antibody protein/kg body weight); 4 hours thereafter an intravitreous injection of ovalbumin was made (0.3-0.4 mg in 75 Ml of sterile saline following passage through a 0.22-IA filter; Nalge Sybron, Rochester, NY). The reaction was evaluated by slit lamp biomicroscopy and achieved a maximum by 20 hours. OAS-2 hour measurements were determined, and histopathologic studies were made at 20 hours after antigen injection. Polymorphonuclear Leukocyte Depletion

Polymorphonuclear leukocytes were depleted with nitrogen mustard (mechlorethamine; 1.75 mg/kg) injected intravenously 3 days prior to intravitreous LPS.'7 White cell and platelet counts were done by standard techniques, and blood smears were used for determining the proportion of PMNs just prior to LPS injection. White blood cell counts were consistently less than 1000 cells/jl, and the proportion of PMNs was less than 5%. Platelet counts were consistently greater than

200,00011A. Complement Depletion Cobra venom factor (CVF; isolated from Naja haja) was used to deplete the third component of complement. Both a commercial source (Cordis Laboratories, Miami, Fla, Lot R4122) and CVF purified by one of us18 were used and gave similar results. Three to four hundred units were given per 2.0 kg rabbit and administered 7 hours before intravitreous LPS. The effects of depletion were measured at 7 and 27 hours after CVF. At the start of these studies, hemolytic complement activity was measured, and throughout these studies serum chemotactic activity was evaluated. CH'50 measurements were performed with a microtiter assay.19 Chemotaxis Assays and Neutrophil Isolation Serum chemotactic activity was induced by incubating serum with zymosan (ICN Chemicals; 1 mg/ml) for 15 minutes at 37 C. Activated serum was diluted in Hanks' buffered salt solution (BSS) 1/100 for use in the lower compartment of a modified Boyden chamber. Chemotactic activity induced by zymosan was compared in serum drawn before CVF and at seven hours and 27 hours after CVF by the leading front technique.20 For all chemotaxis assays, PMNs were suspended in BSS plus 207o bovine serum albumin (BSA) at 2.5 x 106 cells/ml. PMNs were incubated with the putative chemoattractant in the Boyden chamber for 35 minutes at 37 C. The chamber was divided by a 3.0-,I pore-

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VASCULAR PERMEABILITY INDUCED BY LPS

diameter nitrocellulose filter (Sartorius, San Francisco, Calif). Results are expressed as the distance at least two cells migrated into the filter in response to the chemoattractant minus the migration in response to buffer. CH'50 and chemotactic activity were consistently < 100/o of starting levels at the time of LPS injection. For measurements of aqueous chemotactic activity, 0.1-0.20 ml of aqueous was drawn into a 1.0-ml syringe containing EDTA such that the final concentration was 5-10 mM. Assays on aqueous were performed at 20 hours after LPS, and aqueous withdrawn from either the saline-treated control eye or from a normal eye served as a baseline activity. For the chemotactic assays, rabbitYPMNs were isolated from peripheral blood drawn from the central ear artery. Thirty-four milliliters of blood was mixed with 6 ml of acid/citrate/dextrose anticoagulant. Eight milliliters of 60o dextran (wt/vol) T-500 (Pharmacia, Piscataway, NJ) in normal saline was added to the blood, and supernatant was removed after 30 minutes of sedimentation. Supernatant was layered over 56%o Percoll (Sigma) and centrifuged at 450g for 20 minutes. The plasma, Percoll, and mononuclear cell layers were discarded; and the PMN/erythrocyte pellet was suspended in 0.83%0o NH4Cl (wt/vol), pH 7.2, for 7 minutes. Following red blood cell lysis, these PMNs were centrifuged for 10 minutes at 150 g, the pellet washed once and then suspended in BSS containing 2%o BSA. This method yielded greater than 9507o PMNs with excellent viability, as judged by trypan blue exclusion. Human PMNs were isolated by dextran sedimentation and hypotonic lysis.21 Partially purified rabbit C5a was obtained by activating rabbit serum complement by yeast cell wall in the presence of 1 M £-aminocaproic acid. C5a was partially purified by Sephadex and ion exchange chromatography as previously described. 19 This preparation of C5a

Table 2-Effect of Complement Depletion on the Passive Arthus Reaction 2-Hour ocular albumin space (jI/g ± SD) Left eye Right eye (control) (experimental)

Table 1 -Intravitreal LPS Effect on Ocular Vascular Permeability 20 Hours aft(er LPS 2-Hour ocular;albumin space (jA/g

Right eye (LPS)

±L

SD)

,

Left eye

(saline)

PMN depletion

Nitrogen-mustard-treated (7) Normal rabbits (6) Complement depletion (CVF) Complement-depleted (5) Normal rabbits (5) Results

are

expressed

as mean

69.2 + 31.4 21.2 50.7

11.6 11.0

52.1 + 11.9 40.5 6.2

11.1 + 1.0 10.9 + 1.2

SD.

1.7 0.5

Complement depletion (6) Normal rabbits (6)

16.9 ± 7.3* 26.2 + 4.1

10.9 ± 1.2 10.9 ± 1.2

* 0.02 > P > 0.01 relative to permeability induced by the passive Arthus reaction in otherwise normal rabbits.

induced optimal migration in the range of 0.5-1.0% (vol/vol). Stimulus specific desensitization studies were performed with the partially purified rabbit C5a.22 For this purpose, rabbit PMNs (3 x 10/ml) were incubated with 0.5%o C5a for 15 minutes at 4 C. The cells were then washed twice and suspended in BSS plus 2%o BSA for chemotaxis. This method was selected because it markedly reduced subsequent C5a responsiveness without affecting responses to another chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP, Peninsula Laboratories, San Carlos, Calif).

Results Effect of PMN and Complement Depletion on Permeability Measurements of ocular vascular permeability are given in Table 1. All measurements were 20 hours after intravitreal LPS and 2 hours after I2'l-albumin. The LPS-treated eyes of rabbits pretreated with nitrogen mustard had a somewhat higher average OAS than those of control rabbits (69.2 ± 31.4 AId/g, n = 7 versus 50.7 + 21.2 ,.l/g, n = 6, mean + SD). Averages of salinetreated or untreated eyes (not shown) were similar in all groups studied. The LPS-treated eyes of rabbits pretreated with CVF had an average OAS that was similar to that of control rabbits (52.1 ± 11.9 ,ld/g, n = 5 versus 40.5 ± 6.2 Ml/g, n = 5, mean ± SD) (see Table 1). In contrast to its inability to affect the OAS induced by intravitreous LPS, CVF did reduce permeability associated with the passive Arthus reaction. The effect of CVF pretreatment on an ocular passive Arthus reaction is given in Table 2. In pretreated rabbits the average OAS-2 hours (mean ± SD) was 16.9 ± 7.3 (6 rabbits), compared with 26.2 ± 4.1 (6 rabbits) in untreated rabbits. The difference between the treated and untreated groups was significant (0.02 > P > 0.01), even though one of the six pretreated rabbits had an OAS similar to the controls, 29.5 1Au/g. This particular rabbit

38

HOWES ET AL

AJP - January 1985

I

2

'' A

x

3

.

u.

N.

Figure 1-Twenty hours after intravitreous LPS of E coli 055:B5 (0.20 pg/50 Ml). The ciliary processes are edematous, and inflammatory cells are found within and on the surface of processes. The dark blotches in the processes are hemorrhages. (2.5-p section, H&E, x 50) Figure 2-Twenty hours after intravitreous LPS of E coli 055:B5 (0.20 pAgI50 l). Most of the inflammatory cells are polymorphonuclear leukocytes within and on the surface of a ciliary process. (2.0-fA section, H&E, x450) Figure 3-Twenty hours after intravitreous LPS of Ecoli055:B5 (0.20 pg/S5O p) in a rabbit made neutropenic by nitrogen mustard. Processes are edematous, but inflammatory cells and small hemorrhages are absent. (H&E, x 50)

Vol. 118 * No. 1


39

5

4

Figure 4-Twenty hours after intravitreous LPS of E coli 055:B5 (0.20 j4g/50 pal) in a rabbit depleted of C'3 with CVF. Many inflammatory cells are present Figure 5-Twenty hours after intravitreous LPS of E coli 055:B5 (0.20 M4g/50 JA) in a rabbit depleted of over tips of ciliary processes. (H&E, x 200) C'3 with CVF. Most of the inflammatory cells are mononuclear phagocytes. (H&E, x 600)

did appear to be complement-depleted on the basis of the relative inability of zymosan to induce serum chemotactic activity after CVF treatment.

Histologic Changes Typical histopathologic changes 20 hours after intravitreous LPS are shown in Figures 1 and 2. The ciliary processes were edematous and hemorrhagic, and numerous inflammatory cells were seen both within the processes and in the posterior chamber. Most of the cells were PMNs (Figure 2). By contrast, in nitrogenmustard-treated rabbits, edema of the ciliary processes was evident, but no cells were found except for a few macrophages. Hemorrhages were also absent (Figure 3). In CVF-treated rabbits, many cells were present over the ciliary processes, but most of the cells were mononuclear phagocytes (Figures 4 and 5). An estimate of proportion of inflammatory cell types over and within ciliary processes showed that 20 hours after LPS in normal rabbits an average of 62.5 ± 17.3% of cells were PMNs, with a range of 37-930o. By contrast, in CVF-treated rabbits less than 30% of inflammatory cells were PMNs, suggesting that the reduced proportion of PMNs was possibly an effect of the CVF pretreatment.

Histopathologic studies of the passive Arthus reaction in CVF-treated rabbits also disclosed a decrease in edema of the ciliary processes and in the numbers of infiltrating PMNs, compared with untreated rabbits, with the exception of the one rabbit with a higher OAS. Analysis of Chemotactic Activity Aqueous chemotactic activity was measured 20 hours after intravitreous LPS injection in CVF-pretreated and normal rabbits. At 1lo concentration, the aqueous from CVF-treated rabbits showed as much chemotactic activity (15.6 ± 3.0 ,u/35 min [n= 8]) as that from normal rabbits (14.3 ± 3.4 ,/35 min [n=121). (Values represent migration in response to stimulus minus migration in response to buffer alone.) By comparison, relative to normal serum obtained prior to CVF injection, serum from CVF-treated rabbits showed a greater than 90% decrease in chemotactic activity that could be induced by zymosan. To rule out the possibility that aqueous humor induced stimulated random migration (chemokinesis, as opposed to chemotaxis) because of a high aqueous protein concentration,23 a "checkerboard" analysis was performed. For this purpose aqueous in various concentrations was placed both above

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HOWES ET AL

AJP January 0

Table 3-Checkerboard Analysis of Aqueout s-20 Hours After LPS Net migratio n (u/35 min) stimulus albove filter 2 1 0 % Aquenus

1.8 1.8 0.4 15.6 34.8 2 Study using a single aqueous humor representativre of two studies.

Stimulus below filter

0 1

20.0 32.4

115.2

and below the filter in the Boyden chami )er. Maximal migration was induced in the presence of a stimulus gradient, which indicates a true chemotaxi s23 (Table 3). Because chemotactic activity could be the result of complement components derived from complement synthesis within the eye, aqueous chemot actic activity was further analyzed. Twenty hours after intravitreous LPS, the aqueous chemotactic activity ;was found to be partially heat-labile (56 C x 30 minuteDs, net migration, 15.9 ± 5.0, compared with 8.6 ± 5 .3 after heating, n = 7 pairs, mean ± SE); failed to alttract human PMNs at concentrations chemotactic for rabbit PMNs (net migration, 4.8 ± 6.7, n = 3, mean + SD); and was unaffected by prior exposure of the irabbit PMNs to C5a. In fact, preexposure of the PMNs t o C5a tended to increase responsiveness (net migratio n, 5.7 ± 4.4 ,i/35 min, n = 4, mean ± SE) for cells preincubated in buffer; net migration, 12.6 ± 2.5 ,/35 min for cells preincubated in C5a). In the present studlies as well as in studies we have previously reported, pireincubation of PMNs with CSa reduced responsivene.ss to C5a approximately 45' 7 without affecting responLses to FMLP. All of these findings are in contrast to the chemotactic activity of C5a itself and to the chemot,actic activity in aqueous from rabbits given intravenc)us LPS (2.5 jig/kg, Salmonella typhimurium). In the laLtter instance, aqueous contains chemotactic activity antiigenically and biochemically similar to C5a (Table 4).24 Specifically, chemotactic activity in aqueous humor E3 hours after lnrav this dose and preparation of intravenous IL,KrPRa 15ic iaig,sly heat-stable, attracts human PMNs, and is markedly inhibited by prior exposure of PMNs to Cs 5a. These findings strongly support the concept th;at CSa is not the chemotactic activity present in the aqiueous of rabbits following intravitreous LPS injecticin.

Discussion Neither depletion of leukocytes nor systemic depletion of complement affects the alteration in ocular vascular permeability induced by the intravitreous injection of bacterial LPS. In contrast, CVF pretreatment significantly decreased OVP in an ocular passive Arthus reaction. Histopathologic studies of eyes of CVF-

1985

pretreated rabbits showed a decreased proportion of polymorphonuclear leukocytes in the inflammatory infiltrate, compared with controls. Aqueous samples 20 hours after intravitreous LPS in CVF-treated and control rabbits contain chemotactic activity of similar magnitude which does not seem to be related to C5a. Nitrogen mustard depletion of leukocytes was unexpectedly ineffective in preventing an alteration in ocular vascular permeability in the present study. PMN leukocytes have been found to contribute to alterations in vascular permeability in other models of experimental inflammation,4-6 although some of these findings may be attributable to differences in species, target organ, and/or methods of administration of LPS.6 Lysosomal enzymes released during acute or prolonged inflammation, particularly elastase and collagenase, may affect the integrity of basement membranes in blood vessel walls.25 PMN leukocytes can apparently respond to chemical mediators in tissues and influence alterations in vascular permeability. Neutrophil chemotactic factors such as C5a, leukotriene B4, and FMLP injected with prostaglandin E in rabbit skin induce an alteration in vascular permeability which can be prevented by nitrogen mustard depletion of PMN leukocytes.4 Finally, formalin-killed intact E coli organisms injected in the skin of rabbits produce an edema and acute inflammation which is largely prevented by nitrogen mustard pretreatment.5 Two of the aforementioned studies were performed in the skin, perhaps in part accounting for differences in results from the present study. The skin is itself a highly specialized structure, and differences in inflammatory responses from other tissues have been noted.26 The inflammatory process in the eye may also have manifestations unique to that organ. For instance, ocular blood vessels seem particularly sensitive to eicosanoid metabolites. The release of prostaglandins PGE2 Table 4-Comparison of Aqueous Chemotactic Activity After Intravitreous and Intravenous LPS

Intravitreous LPS Aqueous Chemotactic activity LPS (0.1 jig) (20 hours after LPS)* Attracts rabbit PMNs Attracts human PMNs Heat-stable Inhibited by prior exposure to C5a Antigenically related to C5a Altered by systemic

Intravenous LPS Aqueous Chemotactic

activity LPS (2.5 j.g/kg) (3 hours after LPS)

Yes No No No

Yes Yes Yes Yes

Not tested

Yes

No

Not tested

decomplementation *

Times chosen are at the point of maximum clinical inflammation.

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and PGFZ,, has been demonstrated to be an important aspect of experimental and clinical ocular inflammation.2'28 In fact, the trauma resulting from aspiration of aqueous induces an alteration in OVP that is in large measure due to prostaglandin release.29'30 Several studies have suggested that mediators may act synergistically with prostaglandins.4'31'32 The release of neurotransmitter substances such as substance P may also be particularly important in ocular inflammation.33 Although the vascular permeability induced by local LPS was not altered in leukopenic rabbits, hemorrhages of ciliary processes were sharply reduced, compared with those in control rabbits. This finding suggests that LPS of itself does not cause vascular damage of sufficient magnitude to produce hemorrhages. It is compatible with the hypothesis that release of enzymes from leukocytes may be important in determining vascular damage, as suggested by Kopaniak and Movat.5 Interpreting the observation that systemic decomplementation fails to reduce LPS-induced increase in OVP is complex because 1) systemic decomplementation is virtually never 1000o complete, 2) systemic decomplementation does not guarantee the absence of ocular complement, and 3) complement could influence vascular permeability directly as an anaphylatoxin or indirectly as a chemoatttractant. CVF as prepared and used in the present study produced a greater than 90Wo decrease in hemolytic complement activity and in zymosan-inducible chemotactic activity, but direct measurements of complement metabolites, particularly anaphylatoxins, were not carried out. Conceivably, C3a, C4a, and C5a may be important in this regard.34 It is also possible that complement activity normally present in ocular fluids and tissues35'36 is retained after CVF treatment, and/or that activated macrophages may be producing complement locally.37 In a recent study in rats, CVF pretreatment did not prevent ocular changes resulting from intraperitoneal or intravitreous LPS injection, but the authors attributed these results to incomplete local decomplementation.3 It is noteworthy, however, that CVF as used in the present study did cause a decrease in altered ocular vascular permeability in an ocular passive Arthus reaction and a decrease in the proportion of accumulated PMN leukocytes in uveal tissue and the posterior chamber, suggesting some effect on PMN leukocyte chemotaxis. It is also to be noted that CVF treatment itself did not cause an alteration in OVP, suggesting that complement activation alone is not sufficient to cause an alteration in OVP. This lack of effect is similar to that described in the rabbit lung following intravenous infusion of complement-derived factors. 19 Because complement might be playing a role in the local ocular response to LPS in rabbits systemically depleted of complement, aqueous fluids were examined

for evidence of chemotactic activity, most particularly C5-derived peptides, the chemoattractants primarily generated when complement is activated.38 Chemotactic activity was found in the aqueous of both CVF and control rabbits 20 hours after intravitreous LPS. The activity was of similar magnitude in both groups. Intravenous LPS can induce chemotactic activity in aqueous that functionally and biochemically resembles C5a24; but unlike the chemotactic activity induced by intravenous LPS, aqueous chemotactic activity after intravitreous LPS as tested in the present study is partially heat-labile, fails to attract human PMN leukocytes, and most importantly is unaffected by prior exposure of rabbit PMNs to C5a (Table 4). Thus, specific desensitization of rabbit PMNs to rabbit C5a did not affect aqueous chemotactic activity or the response to FMLP, but it did prevent chemoattraction to C5a itself. Results of the present study may simply be a question of timing. If aqueous were examined at an earlier time, perhaps CSa activity might be found; but the reaction is maximal at 20-24 hours, the time examined. Similarly, our results cannot exclude the possibility that a different dose or preparation of LPS would be more dependent on PMNs and/or complement for the induction of ocular vascular permeability. In summary, neither leukocyte depletion with nitrogen mustard nor CVF depletion of complement were effective in preventing the ocular vascular permeability induced by intravitreous LPS. Aqueous chemotactic activity that is not C5a is generated by intravitreous LPS. Other mediators of potential importance in this system include: eicosanoid metabolites, particularly lipoxygenase products such as leukotriene B4,39 plateletactivating factor,40 mononuclear-phagocyte-derived factors such as interleukin-1,41 and fibrin-derived peptides.42

References 1. Sanders TE: The ocular Shwartzman phenomenon. Am J Ophthalmol 1939, 22:1071-1082 2. Bito LZ: The effects of experimental uveitis on anterior uveal prostaglandin transport and aqueous humor composition. Invest Ophthalmol 1974, 13:959-966 3. Bhattacherjee P, Williams RN, Eakins KE: An evaluation of ocular inflammation following the injection of bacterial endotoxin into the rat foot pad. Invest Ophthalmol 1983, 24:196-202 4. Wedmore CV, Williams TJ: Control of vascular permeability by polymorphonuclear leukocytes in inflammation. Nature 1981, 289:646-650 5. Kopaniak MM, Movat HZ: Kinetics of acute inflammation induced by Escherichia coli in rabbits: II. The effect of hyperimmunization, complement depletion, and depletion of leukocytes. Am J Pathol 1983, 110:13-29 6. Heflin AC Jr, Brigham KL: Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J Clin Invest 1981, 68:1253-1260 7. Bito LZ: Inflammatory effects of endotoxin-like con-

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8. 9. 10.

11.

12.

13. 14. 15. 16. 17. 18.

19.

20.

21.

22.

23.

24. 25. 26.

HOWES ET AL

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Acknowledgments The authors wish to acknowledge the technical assistance of Christian Fahlman and Virginia Cruse. Jo Anne Bowman prepared the manuscript.