Leak Syndrome and Perforin in the Induction of Vascular Evidence For

Evidence for the Involvement of Fas Ligand and Perforin in the Induction of Vascular Leak Syndrome

This information is current as Asimah Q. Rafi, Ahmet Zeytun, Michael J. Bradley, D. of October 1, 2021. Phillip Sponenberg, Randolph L. Grayson, Mitzi Nagarkatti and Prakash S. Nagarkatti J Immunol 1998; 161:3077-3086; ; http://www.jimmunol.org/content/161/6/3077 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Evidence for the Involvement of Fas Ligand and Perforin in the Induction of Vascular Leak Syndrome1

Asimah Q. Raﬁ,2* Ahmet Zeytun,* Michael J. Bradley,* D. Phillip Sponenberg,† Randolph L. Grayson,‡ Mitzi Nagarkatti,† and Prakash S. Nagarkatti3*

Endothelial cell injury resulting in vascular leak syndrome (VLS) is one of the most widely noted phenomenons in a variety of clinical diseases. In the current study we used IL-2-induced VLS as a model to investigate the role of cytolytic lymphocytes in the cytotoxicity of endothelial cells. Administration of IL-2 (75,000 U/mouse, three times a day for 3 days) into BL/6 wild-type mice triggered significant VLS in the lungs, liver, and spleen. Interestingly, perforin-knockout (KO) mice exhibited a marked decrease in IL-2-induced VLS in all three organs tested. Also, Fas ligand-defective (gld) mice and Fas-deficient (lpr) mice exhibited decreased VLS in the liver and spleen, but not in the lungs. The decreased VLS seen in perforin-KO, gld, and lpr mice was not due to any defect in lymphocyte migration or homing to various organs because histopathologic studies in these mice demonstrated Downloaded from significant and often greater perivascular infiltration of lymphocytes compared with the IL-2-treated wild-type mice. Ultrastruc- tural studies of the lungs demonstrated significant damage to the endothelial cells in IL-2-treated wild-type mice and decreased damage in perforin-KO mice. IL-2 administration caused up-regulation of CD44 in all strains of mice tested and triggered increased LAK activity against an endothelial cell line in wild-type and gld mice, but not in perforin-KO mice. The current study demonstrates for the first time that perforin and Fas ligand may actively participate in endothelial cell injury and induction of VLS

in a variety of organs. The Journal of Immunology, 1998, 161: 3077–3086. http://www.jimmunol.org/

t sites of chronic inflammation as seen in a variety of and pulmonary edema (8, 9). Such toxicity results from increased infections, autoimmune diseases, graft-vs-host disease, capillary leak, also known as vascular leak syndrome (VLS).3 A A and during treatment of cancer patients with high doses number of cytokines used as hemopoietic growth factors are also of IL-2, significant damage to the endothelial cells has been known known to trigger VLS (9). The exact mechanism of cytokine-trig- to occur, which leads to severe toxicity or pathogenesis associated gered endothelial cell damage and induction of VLS is not clear. with the disease. For example, in murine lymphocytic choriomen- Several recent studies have suggested that VLS may result from ingitis viral infection, massive delayed-type hypersensitivity reac- actual damage to the endothelial cells caused by cytotoxic lym- tion occurs in the cerebrospinal fluid. It has been speculated that phocytes (10, 11). In contrast, some types of endothelial cell dam- by guest on October 1, 2021 virally activated CD8ϩ T cells kill the endothelial cells, leading to age may result from participation of neutrophils and complement massive extravasation of monocytes and CD4ϩ T cells via the components (12). The fact that CTL are involved in the induction subarachnoid space (1). In multiple sclerosis, damage to the blood- of VLS was demonstrated in a recent study from our laboratory in brain barrier has been known to occur after injury to the endothe- which administration of a CTL clone into a syngeneic irradiated lial cells by cytotoxic T cells (2). Also, in autoimmune disease mouse along with IL-2 led to a significant induction of VLS in models involving vasculitides, the lesions have been associated vivo (13). with infiltration of lymphocytes and macrophages at the vascular In the current study we used VLS as a model to study the en- wall structure (3). Such types of vasculitis have been described in dothelial cell damage seen after IL-2 administration. To directly human and murine autoimmune diseases (4, 5). Similarly, in arte- test the involvement of cytotoxic lymphocytes in endothelial cell riosclerosis, endothelial cell damage and inflammatory cell activa- injury leading to the induction of VLS, we used perforin-knockout tion have been shown to contribute to the further development of (KO) and Fas ligand (FasL)-defective, gld mice, inasmuch as per- the cardiovascular disease (6, 7). forin and FasL have been characterized as two important effector Although IL-2 therapy has yielded encouraging results in the molecules involved in cytotoxicity mediated by CTL and NK/LAK treatment of certain types of cancer, its use is limited by dose- cells (14). The results demonstrated that the VLS was markedly dependent toxicity characterized by weight gain, dyspnea, ascites, decreased in all organs tested in perforin-KO mice and was significantly decreased in the liver and spleen of gld mice, thereby supporting the hypothesis that VLS results from direct cytotoxicity Departments of *Biology, ‡Plant Pathology, Physiology, and Weed Science, and †Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary of endothelial cells by cytotoxic lymphocytes. Medicine, Virginia Tech, Blacksburg, VA 24061 Received for publication December 5, 1997. Accepted for publication May 11, 1998. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page Mice charges. This article must therefore be hereby marked advertisement in accordance Four to six-week-old female C57BL/6 mice were purchased from the Na- with 18 U.S.C. Section 1734 solely to indicate this fact. tional Institutes of Health (Bethesda, MD). Age-matched B6/gld/gld B6/ 1 This work was supported in part by grants from the American Cancer Society (IM lpr/lpr and perforin-KO mice were bred in our animal facilities. The per- 747) and the National Institutes of Health (AI101392 and HL058641), and by a Gina forin-KO mice were provided by W. K. Clark (University of California, Finzi Memorial Student Fellowship from the Lupus Foundation of America (to A.R.). 2 Address correspondence and reprint requests to Dr. Prakash S. Nagarkatti, Depart- ment of Biology, Virginia Tech, Blacksburg, VA 24061. E-mail address: 3 Abbreviations used in this paper: VLS, vascular leak syndrome; KO, knockout; [email protected] FasL, Fas ligand; PE, phycoerythrin.

Los Angeles, CA) (15). It should be noted that gld and lpr mice used at 4 Histology to 6 wk of age did not exhibit any lymphoproliferative disease. For histopathologic studies, groups of five separate mice were injected with Cell lines IL-2 or PBS as described above, and on day 4, lungs and liver were fixed in 10% formalin solution. The organs were embedded in paraffin, sec- YAC1, a NK-sensitive Moloney virus-induced lymphoma, and TME-3H3, tioned, and stained with hematoxylin and eosin. Perivascular infiltration an SV40-transformed endothelial cell line, were maintained in vitro by was scaled by counting the number of lymphocytes infiltrating the vessel serial passages, as previously described (13, 16). and averaging the minimum and maximum ranges for each group. Three samples were used for lung, and 10 samples were used for the liver. Antibodies Electron microscopy studies Monoclonal MEL-14 (lymphocyte homing receptor; rat IgG) and anti- Tissue samples were fixed in 5% glutaraldehyde/4.4% formaldehyde/ LFA-1 (M17/4; rat IgG) were grown in vitro as described previously (17). 2.75% picric acid in 0.05 M sodium cacodylate buffer, pH 7.4; washed in The FITC-CD3, PE-CD44, PE-CD8, FITC-CD4, and Jo2 (anti-Fas) mAbs a sodium cacodylate buffer; postfixed in osmium tetroxide; embedded in were purchased from PharMingen (San Diego, CA). FITC-conjugated Polybed 812 resin (Polysciences, Warrington, PA); and studied with an F(abЈ) goat anti-Syrian hamster IgG was purchased from Jackson Immu- 2 electron microscope. noResearch Laboratories (West Grove, PA). [125I]BSA was purchased from ICN (Costa Mesa, CA). Statistical analysis ILs The VLS data in different strains of mice were compared using analysis of variance, and p Ͻ 0.05 was considered statistically significant. Recombinant IL-2 was provided by Hoffmann-La Roche (Nutley, NJ) and Dr. C. Reynolds (National Institutes of Health). Results VLS induction in wild-type, perforin-KO, FasL-defective (gld), Downloaded from Detection of surface molecules using immunofluorescence and Fas-deficient (lpr) mice analysis To directly test whether VLS seen following injection of IL-2 is Splenic T cells and LN cells were analyzed for CD3 and CD44 expression caused by cytolytic effector molecules such as perforin and FasL, by staining the cells with FITC-CD3- and PE-CD44-labeled Abs for 30 min on ice followed by washing three times. LFA-1 and MEL-14 expres- we used perforin-KO and FasL-defective (gld) mice with B/6 sion was detected by staining the cells with unlabeled primary Abs against background and compared the degree of VLS seen with IL-2- these markers for 30 min on ice followed by washing three times. After treated wild-type mice. To this end, groups of five mice received http://www.jimmunol.org/ washing, FITC-conjugated secondary Ab was added to detect the presence 75,000 U of IL-2 three times daily for 3 days and once on day 4. of LFA-1 and Mel-14. The secondary Ab consisted of FITC-conjugated 125 anti-rat IgG F(abЈ) (Cappel Laboratories, Durham, NC). Negative controls On the last day, the mice were injected with [ I]BSA, and VLS 2 125 consisted of fluorescence obtained by staining cells with FITC-conjugated was studied by determining the extravasation of [ I]BSA in the secondary Ab. Fas expression was detected by staining the cells with Jo2 lungs, liver, and spleen. The data on VLS seen in each organ were mAbs (anti-Fas) for 30 min on ice followed by washing three times. After expressed as the mean percent increase in radioactive counts per washing, FITC-conjugated F(abЈ) goat anti-Syrian hamster IgG was added 2 minute in IL-2-treated groups compared with that in their respec- to detect for the presence of Fas. Negative controls consisted of fluorescence obtained by staining cells with FITC-conjugated secondary Ab. The tive controls that received PBS alone as described in Materials and

cells were washed twice and analyzed flow cytometrically. CD3, CD4, Methods. For example in one experiment, the lungs of PBS-treated by guest on October 1, 2021 CD44, and CD8 expression was detected by staining the cells with fluo- controls exhibited 4,614 mean cpm, while the lungs from IL-2- rescein-labeled Ab against these markers for 30 min on ice followed by treated mice showed 10,012 mean cpm. Thus, the percent increase washing three times. Nonspecific staining was blocked by incubation of cells with 0.5% normal mouse serum for 30 min before staining with la- in vascular leak was considered to be 117. A similar approach was beled Ab. Next, 10,000 cells were analyzed by a flow cytometer (EPICS V, used to calculate the degree of VLS in each of the five mice per model 752; Coulter, Miami, FL). group, and the mean percent increase in VLS Ϯ SEM was plotted (Fig. 1) for each organ and each strain of mouse. Quantitation of VLS Figure 1 depicts a representative experiment in which the wild- ϩ ϩ Vascular leak was studied by measuring the extravasation of [125I]BSA type (B/6 / ) mice exhibited significant VLS following IL-2 in- into various organs as described previously (18). Groups of five mice were jection in all three organs tested compared with PBS-treated con- injected i.p. with 75,000 U rIL-2 or PBS as a control, three times a day for trols. Interestingly, the VLS induction varied in the perforin-KO 3 days. On day 4, they received one injection in the morning and 2 h later were injected i.v. with 0.5 ␮Ci of [125I]BSA in 0.5 ml of PBS. After 2 h, and FasL mutant mice. In the lungs, VLS was markedly reduced in the mice were bled to death under anesthesia, and the heart was perfused perforin-KO mice, whereas gld mice did not exhibit any decrease, with heparin in PBS as described previously (18). The lungs, liver, and but, in fact, demonstrated a significant increase in VLS. These data spleen were harvested, placed in vials, and measured in a gamma counter. suggested that perforin, but not FasL, played a key role in VLS The VLS seen in IL-2-treated mice was expressed as the percent increase induction in the lungs. The fact that VLS in the lungs of Fas- in extravasation compared with that in the PBS-treated controls and was calculated as: [(cpm in the organs of IL-2-treated mice Ϫ cpm in the organs deficient (lpr) mice was seen to the same extent as that in the of PBS-treated controls)/(cpm in the organ of PBS treated control)] ϫ 100. wild-type mice further corroborated these results. In the liver, the Each mouse was individually analyzed for vascular leak, and the data perforin-KO, gld, and lpr mice exhibited a significant decrease in Ϯ from five mice were expressed as the mean SEM percent increase in VLS. These data suggested that both perforin and FasL played VLS in IL-2-treated mice compared with that in PBS-treated controls. important roles in VLS induction in the liver. In the spleen, the Cytotoxicity perforin-KO, gld, and lpr mice exhibited decreased levels of VLS compared with the wild-type mice. This decrease was, however, The ability of splenic T cells to lyse various tumor targets was tested using less than that seen in the liver, using similar groups of mice. These 51Cr release assays, described previously (13). Briefly, 5 ϫ 106 target cells 51 experiments were repeated three times with consistent results. The (YAC-1 or TME-3H3) were labeled with NaCrO4 by incubation at 37°C for 1 h. Varying E:T cell ratios in triplicate were added in 96-well round- fact that perforin-KO mice exhibited decreased VLS in all three bottom plates (Falcon 3910, Becton Dickinson, Lincoln Park, NJ) and in- organs tested, indicated that perforin may constitute an important cubated for4hat37°C. Spontaneous release was measured by incubating factor involved in the induction of VLS. In contrast, FasL-based the 51Cr-labeled targets alone, and total release was determined by incubating the labeled target cells with 0.1 M SDS. The supernatants were lytic activity appeared to play a key role in the induction of VLS harvested after 4 h, and radioactivity was measured with a gamma counter in liver and spleen but not lungs. This was further corroborated by (TmAnalytic, Elk Grove Village, IL). the fact that VLS was decreased in the liver and spleen of lpr mice. The Journal of Immunology 3079

the livers of perforin-KO, lpr, and gld mice resulted from decreased migration/homing and ability to cause perivascular inﬁl- tration. These data also suggested that the decreased VLS in the lung, liver, and spleen in perforin-KO mice and the decreased VLS in the liver and spleen of gld and lpr mice may have resulted from the inability of cytolytic lymphocytes to mediate lysis of endothelial cells due to deﬁciency of perforin, FasL, or Fas, respectively.

Ultrastructural studies on injury to endothelial cells during VLS FIGURE 1. VLS induction in wild-type, perforin-KO, FasL-defective To further corroborate that IL-2-induced VLS resulted from actual (gld), and Fas-deficient (lpr) mice. Groups of five mice of the BL/6 wild- damage to the endothelial cells, ultrastructural studies of the lung type (ϩ/ϩ), perforin-KO (PD), FasL-defective (gld), and Fas-deficient were performed. As shown in Figure 4a, A1, the wild-type mice (lpr) strains received 75,000 U of IL-2 three times daily for 3 days and once injected with PBS (control) displayed normal ultrastructural mor- 125 on day 4. Two hours later, the mice were injected with [ I]BSA, and VLS phology of blood vessels. In contrast the wild-type mice injected 125 was studied by determining the extravasation of [ I]BSA in the lungs, with IL-2 had extensive to almost complete destruction of the en- liver, and spleen. The vertical bars represent the percent increase in VLS Ϯ dothelial cell layer of the blood capillaries as well as epithelial cell SEM seen following IL-2 administration compared with that in the PBS- treated controls, as described in Materials and Methods. The counts for the damage in the alveolar air spaces (Fig. 4a, A2). The perforin KO lungs of ϩ/ϩ, perforin-KO, gld, and lpr mice treated with PBS were mice injected with IL-2 had the least amount of damage to the Downloaded from 4,614 Ϯ 248, 6,604 Ϯ 147, 3,219 Ϯ 277, and 7,585 Ϯ 382, respectively. endothelial cells (Fig. 4b, B2). Many endothelial cells appeared to The counts for the liver of ϩ/ϩ, perforin-KO, gld, and lpr mice treated be morphologically normal, as observed in the controls. The IL- with PBS were 17,120 Ϯ 1,628, 20,818 Ϯ 2,378, 18,140 Ϯ 2,773, and 2-treated lpr mice displayed more damage in the blood vessels, 21,616 Ϯ 656, respectively. The counts for the spleen of ϩ/ϩ, perforin- with shrinkage of endothelial cells away from the basal lamina and KO, gld, and lpr mice treated with PBS were 1,455 Ϯ 22, 2,378 Ϯ 191, extensive deterioration of the endothelial and epithelial cells, Ϯ Ϯ 1,700 217, and 1,874 335, respectively. The VLS data from perforin- which are separated by the basal lamina (Fig. 4b, B3). The most http://www.jimmunol.org/ KO, gld, and lpr mice in each organ were compared with their respective severe destruction of endothelial cells and epithelial cells of the data from BL/6 wild-type (ϩ/ϩ) mice, and statistically significant differ- alveolar air spaces and breakage of the basal lamina occurred in ences were indicated with an asterisk. There was no statistically significant difference in counts of specific organs of ϩ/ϩ, perforin-KO, gld,orlpr the IL-2-treated gld mice. In the lungs of these mice, widespread treated with PBS. areas of cellular debris could be observed (Fig. 4b, B1). In some places the basal lamina was broken, and cell debris extruded into the alveolar air spaces. These data together indicated that IL-2 treatment caused signif- These findings supported our hypothesis that IL-2 may activate icant damage to the endothelial cells in wild-type mice, thereby

LAK cells, which mediate lysis of endothelial cells using per- confirming that the VLS resulted from actual damage to the en- by guest on October 1, 2021 forin and FasL. The involvement of FasL also suggested that dothelial cells. Furthermore, when KO/mutant mice were screened, endothelial cells may express Fas, as previously shown in our the endothelial cell damage correlated with the VLS data. It was laboratory (20). interesting to note that perforin-KO mice exhibited the least damage to the endothelial cells in the lungs. Histopathologic studies of organs exhibiting VLS It was possible that the reason why perforin-KO, gld,orlpr mice LAK activity in mice undergoing VLS exhibited decreased VLS was because of the inability of cytolytic To test whether the lymphocytes from IL-2-treated mice would ex- lymphocytes to migrate to the vascular tissues of various organs hibit increased LAK activity and to investigate the nature of cytolytic rather than the inability to mediate cytotoxicity of endothelial cells. effector molecules triggered by in vivo IL-2 administration, splenic T To rule out this possibility and to confirm that in IL-2-treated mice cells collected from mice undergoing VLS were tested for cytolytic there was perivascular infiltration of mononuclear cells, we con- activity against Fasϩ YAC-1 tumor targets. The data shown in Figure ducted histopathologic studies. To this end, wild-type, perforin- 5(upper panel) indicated that perforin-KO (B) and gld (C) mice ex- KO, gld, and lpr mice were injected with PBS or IL-2 as described hibited minimal spontaneous cytotoxicity against YAC-1 targets. earlier. On day 4, the mice were euthanized, and various organs were fixed, processed, and stained with hematoxylin and eosin. The representative studies on the lungs and liver are shown in Figures 2 and 3, respectively. Table I. Perivascular infiltrating lymphocytes from mice treated with In the lungs and liver, IL-2 treatment led to significant perivas- IL-2 a cular infiltration in all four strains of mice, consisting mainly of lymphocytes (Figs. 2 and 3, A2, B2, C2, and D2), whereas PBS- Strain treated groups failed to exhibit significant lymphocytic infiltration Organ ϩ/ϩ Peforin-KO gld lpr (Figs. 2 and 3, A1, B1, C1, and D1). The degree of infiltration was also measured by counting the number of lymphocytes infiltrating Lungb 3.17 Ϯ 0.12 5.95 Ϯ 0.38* 3.45 Ϯ 0.23 5.00 Ϯ 0.36* c Ϯ Ϯ Ϯ Ϯ each vessel and averaging the range for each group (Table I). Liver 2.80 0.12 3.50 0.063* 2.98 0.25 3.12 0.19 These results showed that IL-2-treated perforin-KO mice had the a Mice were injected with IL-2 to induce VLS and the organs were processed for highest level of perivascular infiltration followed by lpr, gld, and histopathological studies as described in Figures 2 and 3. The degree of perivascular infiltration was measured by counting the number of lymphocytes infiltrating each wild-type mice (Figs. 2 and 3, and Table I). Although the reason vessel. why perforin KO, gld, and lpr mice had higher levels of perivas- b The number of lymphocytes infiltrating a venule. The data represent the mean Ϯ SEM obtained from 3 (for lungs) or 10 (for liver) samples per mouse (4 mice cular infiltration was not clear, these data ruled out the possibility were analyzed for each organ). Data showing statistically significant differences (p that the decreased VLS seen in the lungs of perforin-KO mice and Ͻ 0.05) when compared to the wild-type mice are indicated with an asterisk. 3080 VASCULAR LEAK SYNDROME IN FasL-DEFECTIVE AND PERFORIN-DEFICIENT MICE Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Histopathologic studies on lung in mice. Lungs from wild-type (A1 and A2), perforin-KO (B1 and B2), gld (C1 and C2), and lpr (D1 and D2) mice treated with PBS (A1, B1, C1, and D1) or IL-2 (A2, B2, C2, and D2) were harvested and preserved in 10% formalin solution. Sections were stained with hematoxylin and eosin. Perivascular inﬁltration, consisting mostly of lymphocytes, is indicated by arrows. The 1-cm horizontal bar equals 35 ␮m.

However, after IL-2 administration, they exhibited a signiﬁcant in- regulates both FasL-based and perforin-based cytotoxicity, which crease in cytotoxicity, although such lytic activity was less than that may subsequently play a role in endothelial cell lysis. To test the seen in the wild-type mice (A). These data indicated that IL-2 up- ability of IL-2-induced LAK cells to kill endothelial cells, we used a The Journal of Immunology 3081 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Histopathologic studies on liver in mice. Livers from wild-type (A1 and A2), perforin-KO (B1 and B2), gld (C1 and C2), and lpr (D1 and D2) mice treated with PBS (A1, B1, C1, and D1) or IL-2 (A2, B2, C2, and D2) were harvested and preserved in 10% formalin solution. Sections were stained with hematoxylin and eosin. Perivascular inﬁltration consisting mostly of lymphocytes is indicated by arrows. The 1-cm horizontal bar equals 35 ␮m.

well-characterized endothelial cell line, TME-3H3 (13). The data toxicity of endothelial cells. However, IL-2-induced LAK cells from shown in Figure 5 (lower panels) indicated that wild-type (D), per- wild-type mice mediated the highest level of cytotoxicity, followed by forin-KO (E), and gld (F) mice failed to exhibit spontaneous cyto- those from gld and perforin-KO mice. These data correlated well with 3082 VASCULAR LEAK SYNDROME IN FasL-DEFECTIVE AND PERFORIN-DEFICIENT MICE Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 The Journal of Immunology 3083 Downloaded from

FIGURE 5. LAK activity in mice undergoing VLS against YAC-1 and endothelial cell targets. Wild-type (A and D), perforin-KO (B and E), and gld (C and F) mice were injected with IL-2 as described in Figure 1. On day 4, spleen cells were passed over nylon wool to enrich T cells and were tested for cytotoxicity against 51Cr-labeled YAC-1 targets (A–C) or the endothelial cell line, TME-3H3 (D–F). The data indicate the mean percent cytotoxicity Ϯ

of triplicate cultures SEM. http://www.jimmunol.org/

the VLS results (Fig. 1) in which perforin-KO mice were found to 22). Inasmuch as endothelial cells express the ligand for CD44, we exhibit marked decrease in VLS, and gld mice showed only a partial have hypothesized that LAK cells expressing CD44 may sponta- decrease in certain organs. It should be noted that YAC-1 and TME- neously kill endothelial cells following interaction between LAK 3H3 cell lines expressed significant levels of Fas as determined by cells and endothelial cells (13, 17, 20). We therefore investigated flow cytometry (Fig. 6). However, the endothelial cell line expressed whether IL-2 treatment would up-regulate CD44 expression in T lower levels of Fas, and only 42% of the cells expressed Fas com- cells. To this effect, wild-type, perforin-KO, gld, and lpr mice pared with the thymocytes from wild-type mice, which expressed injected with IL-2 as described before and were sacrificed, and by guest on October 1, 2021 higher levels. This may explain why endothelial cell lysis was purified T cells from the spleens were stained using mAbs against more dependent on perforin than FasL as shown in Figure 5 CD44 and were analyzed flow cytometrically. (lower panel). The data shown in Figure 7 indicated that in IL-2-treated mice, there was significant up-regulation in CD44 expression on T cells Effect of IL-2 administration on CD44 expression in all groups of mice tested compared with that in the controls. We and others have demonstrated that activation through CD44 This was evident from the increase in the mean intensity of fluo- can trigger lytic activity in CTL and NK/LAK cells (16, 17, 21, rescence on T cells following IL-2 administration. It should be

FIGURE 4. a, Ultrastructural studies on injury to endothelial cells during VLS. Lung samples from wild-type mice treated with IL-2 (A2) or PBS (A1) were fixed and studied with an electron microscope. A1 shows wild-type mice treated with PBS. A near cross-section of a capillary has an erythrocyte (E) in the central lumen surrounded by endothelial cells (En) with their cytoplasm tightly appressed against the basal lamina (BL), which separates the endothelial cell from the flattened cytoplasmic portion of a lining epithelial cell. Note the numerous pinocytotic vesicles (PV) and a few tight junctions (TJ). It should be noted that the lung tissue from perforin-KO, gld, and lpr mice treated with PBS alone did not show any damage to the endothelial cells and was structurally similar to that in PBS-treated wild-type mice and therefore is not depicted. Magnification ϭϫ16,000. A2 depicts wild-type mice treated with IL-2. A cross-section of a capillary with erythrocytes (E) in the lumen is shown. Almost complete destruction of the endothelial cell layer has occurred. Epithelial (Ep) cells of the alveolar air space on the other side of the basal lamina (BL) show extensive damage. Magnification ϭϫ6,600. b, Ultrastructural studies on injury to endothelial cells during VLS. Lung samples from gld (B1), perforin-KO (B2), and lpr (B3) mice treated with IL-2 were fixed and studied with an electron microscope. B1 shows gld mice treated with IL-2. Very extensive damage has occurred, resulting in cell death within the blood vessel and outside the basal lamina (BL). The cellular cytoplasm of the endothelial cells and the epithelial cells of the alveolar sac are completely fragmented into cellular debris. In the lumen a white blood cell (WBC) is in the process of being fragmented. The basal lamina (BL) has been broken, with cellular debris spilling into the alveolar air space, including pinocytotic vesicles that are involved in the transport of fluids (arrow). Magnification ϭϫ12,600. B2, shows perforin-KO mice treated with IL-2. Erythrocytes (E) are moving through the lumen of the capillary. The endothelial cells are still pressed against the basal lamina (BL), but show indications of cellular deterioration. This is indicated by the formation of membranous circular structures (arrows) and apoptotic bodies in the endothelial cells. However, this deterioration is much less than that seen in the wild-type mice treated with IL-2. This is based on the intact membranes of the cellular constituents, the intact basal lamina, and the lack of cellular debris and membrane fragments. Magnification, ϭϫ20,400. B3 shows lpr mice treated with IL-2. Cell damage to the endothelial and epithelial cells separated by the basal lamina is evident. Cell organization and structures have been lost, leaving cell remnants. There is condensed disorganized dark-staining cellular debris (large arrows) in the endothelial (En) and epithelial (Ep) cells. The cell blebbing and the loss of cell membranes are indicative of dying and dead cells. The basal lamina (BL) is losing its definition and has become diffuse between the endothelial and epithelial cells, which indicates severe leakage. Magnification ϭϫ50,000. 3084 VASCULAR LEAK SYNDROME IN FasL-DEFECTIVE AND PERFORIN-DEFICIENT MICE

FIGURE 6. Fas expression in YAC-1 and the endothelial cell line TME- 3H3. Cells were stained with Jo2 mAbs against Fas followed by FITC- Ј conjugated F(ab )2 goat anti-Syrian hamster IgG (b). Negative controls (a) Downloaded from Ј consisted of cells stained with FITC-conjugated F(ab )2 goat anti-Syrian hamster IgG alone. The cells were analyzed ﬂow cytometrically.

noted that IL-2 treatment up-regulated the expression of LFA-1

and L-selectin and down-regulated CD3 expression to a moderate http://www.jimmunol.org/ extent in all strains of mice (data not shown). FIGURE 7. Effect of IL-2 administration on CD44 expression. Wild- IL-2 treatment increases the percentage of CD8ϩ T cells and type (ϩ/ϩ), perforin-KO (pd), gld, and lpr mice injected with IL-2, as decreases the proportion of CD4ϩ T cells in the periphery described in Figure 1, were sacrificed, and purified T cells from the spleens ϩ ϩ were stained using PE-conjugated mAbs against CD44 and were analyzed To investigate the effects of IL-2 treatment on CD4 T and CD8 flow cytometrically. The solid histogram represents autofluorescence, and T cells in the periphery and to compare the levels of their induction the broken histogram represents cells stained with PE-anti-CD44. The per- ϩ ϩ in different groups of mice, the proportions of CD4 and CD8 T centage of positive cells expressing CD44 and the mean intensity of flu- cells in the spleens were detected using flow cytometry. The data orescence, shown in parentheses, are depicted in each histogram. shown in Table II indicated that IL-2 treatment caused an increase by guest on October 1, 2021 in the percentage of CD8ϩ T cells and a consequent decrease in the percentage of CD4ϩ T cells in the periphery of wild-type, perforin- KO, gld, and lpr mice. These data indicated that IL-2 induced whereas gld and particularly the perforin-KO mice exhibited a sig- ϩ similar activation of CD8 in all strains of mice tested and that the nificant decrease in their ability to kill endothelial cells. Lympho- differences in cytotoxicity seen in various groups did not result cytes from all strains of mice tested expressed increased levels of from altered activation of T cell subsets. CD8ϩ T cells and higher densities of homing molecules, including CD44, following IL-2 treatment. These data together suggested Discussion that IL-2 up-regulates the expression of CD44, thereby facilitating Administration of IL-2, although promising in the treatment of the migration of LAK cells to various organs. Furthermore, IL-2 certain types of cancer, triggers severe side effects in patients who also up-regulates perforin and FasL, and the interaction between develop a capillary leak syndrome resulting in anasarca and mul- LAK cells and endothelial cells may trigger their lysis, leading to tiorgan system dysfunction (23, 24). The exact mechanism of in- extravasation of intravascular fluid. duction of VLS is not known. In the current study we tested the Earlier studies have demonstrated that immunosuppression of hypothesis that IL-2-induced VLS may result from the cytolytic mice by pretreatment with irradiation or injection of cyclophosph- activity of endothelial cells by LAK cells. Because perforin and amide or cortisone markedly reduces or eliminates the develop- FasL constitute two important cytolytic effector molecules in- ment of IL-2-induced VLS (18). These data suggested that IL-2 volved in LAK cell-mediated cytotoxicity, we used perforin-KO does not act directly on the blood vessels to alter their permeabil- and FasL-mutant (gld) mice to study the VLS. Our data indicated ity, but that it may do so indirectly by involving cells of the im- that IL-2 up-regulates perforin and FasL activity and that these mune system. Vascular leaks in acute inflammatory lesions are molecules do participate in the induction of VLS. In wild-type triggered by mediators of immediate hypersensitivity, such as his- mice exhibiting VLS, there was significant perivascular infiltration tamine, serotonin, and bradykinin (25). Although the IL-2-induced with lymphocytes, and endothelial cell damage was evident from ultrastructural studies. In perforin-KO mice, despite perivascular vascular leak resembles that triggered by the above-mentioned me- infiltration with lymphocytes, there was no significant endothelial diators, earlier studies have ruled out the involvement of vasoac- cell damage, and VLS was markedly reduced in all organs tested. tive amines in IL-2-induced VLS (18). Lymphocytes are also Similarly, the VLS was decreased in certain organs of gld and lpr known to produce a variety of mediators that increase the vascular mice, thereby suggesting that Fas-FasL interactions may also reg- permeability (26, 27). However, the current study demonstrates ulate VLS induction. Also, the IL-2-treated wild-type mice exhib- that IL-2-induced vascular leak may result from the involvement ited increased LAK cell activity against an endothelial cell line, of cytolytic effector molecules, perforin and FasL. The Journal of Immunology 3085

Table II. Expression of CD4 and CD8 in the spleen after IL-2 the liver and spleen suggested that the endothelial cells in these treatment a organs may express Fas. The endothelial cell line used in the current study expressed significant levels of Fas. Whether the endo- PBS IL-2 thelial cells in different organs express varying levels of Fas, Strain CD4 CD8 CD4 CD8 thereby accounting for the differential susceptibility to FasL-based cytotoxicity, remains to be determined. It should be noted that in Ϯ Ϯ Ϯ Ϯ Wild-type 28.6 1.3 14.2 3.7 20.2 0.09* 24.2 1.1* the lungs, perforin, but not FasL, played an important role in VLS PD 21.2 Ϯ 0.18 17.6 Ϯ 0.82 16.87 Ϯ 0.62* 22.4 Ϯ 0.05* GLD 17.1 Ϯ 1.2 15.8 Ϯ 0.62 12.78 Ϯ 0.93* 24.8 Ϯ 0.82* induction. However even in perforin-KO mice the VLS was not LPR 20.7 Ϯ 0.81 19.45 Ϯ 1.9 14.15 Ϯ 0.43* 36.15 Ϯ 1.9* completely abolished. This suggested the possibility that other cytotoxic molecules such as TNF may play a role in VLS induction. a Mice were injected with PBS or IL-2 as described in Figure 1. The spleen cells were stained for CD4 and CD8 markers and cells were analyzed flow cytometrically. It should be noted that the role of TNF in VLS is controversial. The data are depicted as mean percent positive cells Ϯ SEM obtained from four to Studies involving attempts to block TNF have demonstrated either five mice. The CD4ϩ and CD8ϩ T cell percentages in IL-2-treated mice were compared with their respective PBS-treated controls, and those showing statistically sig- beneficial or no effect on IL-2-induced toxicity (32, 33). In addi- nificant differences (p Ͻ 0.05) are indicated with an asterisk. tion, the VLS may result from complement activation (34, 35). In the current investigation the histopathologic studies indicated that following IL-2 administration, there was significant infiltration Several studies have suggested that endothelial cells can be the of lymphocytes, but not neutrophils, in the perivascular tissue. targets of lymphocyte-mediated destruction. Damle et al. demon- These studies ruled out the possible damage to endothelial cells strated that IL-2-activated LAK cells adhered and killed endothe- caused by neutrophils as reported in other models (36–38). The Downloaded from lial cells efficiently (10). Also, the toxicity associated with IL-2 fact that IL-2-treated perforin-KO, gld, and lpr mice demonstrated therapy has been shown to decrease after depletion of NK cells in significant perivascular infiltration of lymphocytes, in fact greater vivo (29). The role of CTL in VLS induction was also demon- than that in the IL-2-treated wild-type mice, ruled out the possi- strated in our earlier studies, in which it was noted that adminis- bility that the decreased VLS seen in the KO/mutant mice was tration of a CTL clone plus IL-2 into irradiated syngeneic mice, but because of the inability of cytolytic lymphocytes to migrate to the not the CTL clone or IL-2 alone, triggered VLS (13). Also, the

vascular tissues of various organs. Also, the fact that all strains http://www.jimmunol.org/ IL-2-activated CTL clone could mediate efficient lysis of an en- tested exhibited similar up-regulation of CD44, a molecule in- dothelial cell line, but not a fibroblast cell line, in an MHC-unre- volved in lymphocyte homing, further suggested that decreased stricted fashion (13). Moreover, methotrexate was found to atten- VLS in the KO/mutant mice was due to the deficiency of the cy- uate pulmonary vascular leak by preventing the proliferation of tolytic effector molecules rather than lymphocyte migration and NK/LAK cells and also by inhibiting leukocyte binding to endo- homing. Our data are consistent with previous histologic studies thelium (30). Similarly, dextran sulfate, which blocks leukocyte- that showed lymphoid cell infiltration in lung, liver, kidney, and endothelial adhesion, attenuates IL-2-induced VLS (31). Such heart following IL-2 therapy (39, 40). Also, the electron micro- studies together with the data presented in the current study sug- scopic studies confirmed that IL-2 treatment in wild-type mice

gest that IL-2-induced VLS may result from the direct cytotoxicity by guest on October 1, 2021 caused significant damage to the endothelial cells, and further- of endothelial cells by LAK cells. more, the results on VLS as seen in various mutant/KO mice cor- In the current study it was observed that perforin-KO mice ex- related well with the ultrastructural studies on endothelial cell hibited marked decrease in VLS in the lungs, whereas gld mice did not exhibit any decrease, but, in fact, exhibited a significant in- damage. It should be noted that the lpr and gld mice used in the crease in VLS in the lungs. These data suggested that perforin current study were 4 to 6 wk of age, during which time they have played a key role in VLS induction in the lungs and that FasL was similar proportions of T cells, as shown in our earlier studies on the not critical. The fact that VLS in Fas-deficient (lpr) mice was of thymus (41). This, however, did not rule out the possibility that the same extent as that in the wild-type mice, further corroborated such differences could exist in the periphery, particularly after IL-2 these results. In the current study it was not clear why the gld mice treatment. In the current study it was noted that IL-2 treatment caused a similar increase in the percentage of CD8ϩ T cells and a exhibited increased VLS in the lungs. This was seen in all three ϩ repeated experiments. It can be speculated that increased perivas- decrease in CD4 T cells in all groups of mice. These data ruled out the possibility that the differences in the cytotoxicity and VLS cular infiltration of lymphocytes as seen in the histopathology of ϩ ϩ lung tissue and/or increased susceptibility of lung endothelial cells induction resulted from differential activation of CD4 or CD8 T to cytolytic activity may have triggered increased VLS in gld mice. cells in various groups of mice. Also, in gld mice, there was greater VLS in the lungs but less in the Although in the current study we used IL-2-induced VLS as a liver and spleen, thereby suggesting more damage to lung endo- model to study LAK cell-endothelial cell interactions, there is thelium and less to the endothelium of spleen and liver. However, growing evidence that similar endothelial cell injury may occur in when LAK cells from gld mice were tested for cytotoxicity against a variety of disease models. Thus, the vascular leak seen at sites of the endothelial cell line in vitro, they exhibited similar levels of chronic inflammation involving mononuclear cells may be trig- cytotoxicity as the wild-type mice. This may be because the en- gered by the direct killing of endothelial cells by cytolytic lym- dothelial cell line used was derived from the lymph node. Thus, it phocytes involving perforin and FasL-based pathways. is possible based on our data that endothelial cells from various We and others have shown earlier that CTL, double-negative T organs, following IL-2 treatment in vivo, may vary in their sus- cells and NK cells upon activation express high levels of CD44 ceptibility to lysis by LAK cells. The fact that in the liver and the and mediate efficient MHC-unrestricted TCR-independent lysis spleen, both the perforin-KO and gld mice had significantly di- following ligation of CD44 (13, 17, 20–22, 42). We have also minished VLS suggested that both FasL and perforin may play a demonstrated that the lysis of endothelial cells ex vivo by cytolytic significant role in VLS induction in these organs. These data also lymphocytes can be blocked by soluble CD44 fusion protein, anti- suggested that the endothelial cells from different organs may ex- CD44 Fab, or soluble hyaluronate (unpublished observations). hibit differential susceptibility to perforin and FasL-mediated cy- These data suggested that CD44-hyaluronate interactions may play totoxicity. The fact that lpr mice also exhibited decreased VLS in an important role in the migration, homing, and lysis of endothelial 3086 VASCULAR LEAK SYNDROME IN FasL-DEFECTIVE AND PERFORIN-DEFICIENT MICE cells. Further studies on the CD44 isoforms involved in lympho- 22. Tan, P. H., E. B. Santos, H. C. Rossbach, and B. M. Sandmaier. 1993. Enhance- cyte adhesion and cytotoxicity of the endothelial cells should pro- ment of natural killer activity by an antibody to CD44. J. Immunol. 150:812. 23. Rosenberg, S., M. T. Lotze, L. M. Muul, S. Leitman, A. E. Chang, vide useful information on therapeutic intervention to prevent en- S. E. 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