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Departments of
*
Biology,
Plant Pathology, Physiology, and Weed Science, and
Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although IL-2 therapy has yielded encouraging results in the treatment of certain types of cancer, its use is limited by dose-dependent toxicity characterized by weight gain, dyspnea, ascites, and pulmonary edema (8, 9). Such toxicity results from increased capillary leak, also known as vascular leak syndrome (VLS).3 A number of cytokines used as hemopoietic growth factors are also known to trigger VLS (9). The exact mechanism of cytokine-triggered endothelial cell damage and induction of VLS is not clear.
Several recent studies have suggested that VLS may result from actual damage to the endothelial cells caused by cytotoxic lymphocytes (10, 11). In contrast, some types of endothelial cell damage may result from participation of neutrophils and complement components (12). The fact that CTL are involved in the induction of VLS was demonstrated in a recent study from our laboratory in which administration of a CTL clone into a syngeneic irradiated mouse along with IL-2 led to a significant induction of VLS in vivo (13).
In the current study we used VLS as a model to study the endothelial cell damage seen after IL-2 administration. To directly test the involvement of cytotoxic lymphocytes in endothelial cell injury leading to the induction of VLS, we used perforin-knockout (KO) and Fas ligand (FasL)-defective, gld mice, inasmuch as perforin and FasL have been characterized as two important effector molecules involved in cytotoxicity mediated by CTL and NK/LAK cells (14). The results demonstrated that the VLS was markedly 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 of endothelial cells by cytotoxic lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four to six-week-old female C57BL/6 mice were purchased from the National Institutes of Health (Bethesda, MD). Age-matched B6/gld/gld B6/lpr/lpr and perforin-KO mice were bred in our animal facilities. The perforin-KO mice were provided by W. K. Clark (University of California, Los Angeles, CA) (15). It should be noted that gld and lpr mice used at 4 to 6 wk of age did not exhibit any lymphoproliferative disease.
Cell lines
YAC1, a NK-sensitive Moloney virus-induced lymphoma, and TME-3H3, an SV40-transformed endothelial cell line, were maintained in vitro by serial passages, as previously described (13, 16).
Antibodies
Monoclonal MEL-14 (lymphocyte homing receptor; rat IgG) and anti-LFA-1 (M17/4; rat IgG) were grown in vitro as described previously (17). The FITC-CD3, PE-CD44, PE-CD8, FITC-CD4, and Jo2 (anti-Fas) mAbs were purchased from PharMingen (San Diego, CA). FITC-conjugated F(ab')2 goat anti-Syrian hamster IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). [125I]BSA was purchased from ICN (Costa Mesa, CA).
ILs
Recombinant IL-2 was provided by Hoffmann-La Roche (Nutley, NJ) and Dr. C. Reynolds (National Institutes of Health).
Detection of surface molecules using immunofluorescence analysis
Splenic T cells and LN cells were analyzed for CD3 and CD44 expression 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 expression was detected by staining the cells with unlabeled primary Abs against these markers for 30 min on ice followed by washing three times. After washing, FITC-conjugated secondary Ab was added to detect the presence of LFA-1 and Mel-14. The secondary Ab consisted of FITC-conjugated anti-rat IgG F(ab')2 (Cappel Laboratories, Durham, NC). Negative controls consisted of fluorescence obtained by staining cells with FITC-conjugated secondary Ab. Fas expression was detected by staining the cells with Jo2 mAbs (anti-Fas) for 30 min on ice followed by washing three times. After washing, FITC-conjugated F(ab')2 goat anti-Syrian hamster IgG was added to detect for the presence of Fas. Negative controls consisted of fluorescence obtained by staining cells with FITC-conjugated secondary Ab. The cells were washed twice and analyzed flow cytometrically. CD3, CD4, CD44, and CD8 expression was detected by staining the cells with fluorescein-labeled Ab against these markers for 30 min on ice followed by washing three times. Nonspecific staining was blocked by incubation of cells with 0.5% normal mouse serum for 30 min before staining with labeled Ab. Next, 10,000 cells were analyzed by a flow cytometer (EPICS V, model 752; Coulter, Miami, FL).
Quantitation of VLS
Vascular leak was studied by measuring the extravasation of [125I]BSA into various organs as described previously (18). Groups of five mice were injected i.p. with 75,000 U rIL-2 or PBS as a control, three times a day for 3 days. On day 4, they received one injection in the morning and 2 h later were injected i.v. with 0.5 µCi of [125I]BSA in 0.5 ml of PBS. After 2 h, the mice were bled to death under anesthesia, and the heart was perfused with heparin in PBS as described previously (18). The lungs, liver, and spleen were harvested, placed in vials, and measured in a gamma counter. The VLS seen in IL-2-treated mice was expressed as the percent increase in extravasation compared with that in the PBS-treated controls and was calculated as: [(cpm in the organs of IL-2-treated mice - cpm in the organs of PBS-treated controls)/(cpm in the organ of PBS treated control)] x 100.
Each mouse was individually analyzed for vascular leak, and the data from five mice were expressed as the mean ± SEM percent increase in VLS in IL-2-treated mice compared with that in PBS-treated controls.
Cytotoxicity
The ability of splenic T cells to lyse various tumor targets was tested using 51Cr release assays, described previously (13). Briefly, 5 x 106 target cells (YAC-1 or TME-3H3) were labeled with 51NaCrO4 by incubation at 37°C for 1 h. Varying E:T cell ratios in triplicate were added in 96-well round-bottom plates (Falcon 3910, Becton Dickinson, Lincoln Park, NJ) and incubated for 4 h at 37°C. Spontaneous release was measured by incubating the 51Cr-labeled targets alone, and total release was determined by incubating the labeled target cells with 0.1 M SDS. The supernatants were harvested after 4 h, and radioactivity was measured with a gamma counter (TmAnalytic, Elk Grove Village, IL).
Histology
For histopathologic studies, groups of five separate mice were injected with 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, sectioned, and stained with hematoxylin and eosin. Perivascular infiltration was scaled by counting the number of lymphocytes infiltrating the vessel and averaging the minimum and maximum ranges for each group. Three samples were used for lung, and 10 samples were used for the liver.
Electron microscopy studies
Tissue samples were fixed in 5% glutaraldehyde/4.4% formaldehyde/2.75% picric acid in 0.05 M sodium cacodylate buffer, pH 7.4; washed in a sodium cacodylate buffer; postfixed in osmium tetroxide; embedded in Polybed 812 resin (Polysciences, Warrington, PA); and studied with an electron microscope.
Statistical analysis
The VLS data in different strains of mice were compared using analysis of variance, and p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To directly test whether VLS seen following injection of IL-2 is
caused by cytolytic effector molecules such as perforin and FasL, we
used perforin-KO and FasL-defective (gld) mice with B/6
background and compared the degree of VLS seen with IL-2-treated
wild-type mice. To this end, groups of five mice received 75,000 U of
IL-2 three times daily for 3 days and once on day 4. On the last day,
the mice were injected with [125I]BSA, and VLS was
studied by determining the extravasation of [125I]BSA in
the lungs, liver, and spleen. The data on VLS seen in each organ were
expressed as the mean percent increase in radioactive counts per minute
in IL-2-treated groups compared with that in their respective controls
that received PBS alone as described in Materials and
Methods. For example in one experiment, the lungs of PBS-treated
controls exhibited 4,614 mean cpm, while the lungs from IL-2-treated
mice showed 10,012 mean cpm. Thus, the percent increase in vascular
leak was considered to be 117. A similar approach was used to calculate
the degree of VLS in each of the five mice per group, and the mean
percent increase in VLS ± SEM was plotted (Fig. 1
) for each organ and each strain of
mouse.
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depicts a representative experiment in which the wild-type
(B/6+/+) mice exhibited significant VLS following IL-2
injection in all three organs tested compared with PBS-treated
controls. Interestingly, the VLS induction varied in the perforin-KO
and FasL mutant mice. In the lungs, VLS was markedly reduced in
perforin-KO mice, whereas gld mice did not exhibit any
decrease, but, in fact, demonstrated a significant increase in VLS.
These data suggested that perforin, but not FasL, played a key role in
VLS induction in the lungs. The fact that VLS in the lungs of
Fas-deficient (lpr) mice was seen to the same extent
as that in the wild-type mice further corroborated these results. In
the liver, the perforin-KO, gld, and lpr mice
exhibited a significant decrease in VLS. These data suggested that both
perforin and FasL played important roles in VLS induction in the liver.
In the spleen, the perforin-KO, gld, and lpr mice
exhibited decreased levels of VLS compared with the wild-type mice.
This decrease was, however, less than that seen in the liver, using
similar groups of mice. These experiments were repeated three times
with consistent results. The fact that perforin-KO mice exhibited
decreased VLS in all three organs tested, indicated that perforin may
constitute an important factor involved in the induction of VLS. In
contrast, FasL-based lytic activity appeared to play a key role in the
induction of VLS in liver and spleen but not lungs. This was further
corroborated by the fact that VLS was decreased in the liver and spleen
of lpr mice. These findings supported our hypothesis
that IL-2 may activate LAK cells, which mediate lysis of endothelial
cells using perforin and FasL. The involvement of FasL also suggested
that endothelial cells may express Fas, as previously shown in our
laboratory (20). Histopathologic studies of organs exhibiting VLS
It was possible that the reason why perforin-KO, gld,
or lpr mice exhibited decreased VLS was because of the
inability of cytolytic lymphocytes to migrate to the vascular tissues
of various organs rather than the inability to mediate cytotoxicity of
endothelial cells. To rule out this possibility and to confirm that in
IL-2-treated mice there was perivascular infiltration of mononuclear
cells, we conducted histopathologic studies. To this end, wild-type,
perforin-KO, gld, and lpr mice were injected with
PBS or IL-2 as described 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.
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and 3, A2, B2,C2, and D2), whereas PBS-treated groups failed to
exhibit significant lymphocytic infiltration (Figs. 2 and 3,
A1, B1, C1, and D1). The
degree of infiltration was also measured by counting the number of
lymphocytes infiltrating each vessel and averaging the range for each
group (Table I). These results showed
that IL-2-treated perforin-KO mice had the highest level of
perivascular infiltration followed by lpr, gld,
and wild-type mice (Figs. 2 and 3, and Table I). Although the reason
why perforin KO, gld, and lpr mice had higher
levels of perivascular infiltration was not clear, these data ruled out
the possibility that the decreased VLS seen in the lungs of perforin-KO
mice and the livers of perforin-KO, lpr, and gld
mice resulted from decreased migration/homing and ability to cause
perivascular infiltration. 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.
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To further corroborate that IL-2-induced VLS resulted from actual
damage to the endothelial cells, ultrastructural studies of the lung
were performed. As shown in Figure 4a, A1, the
wild-type mice injected with PBS (control) displayed normal
ultrastructural morphology of blood vessels. In contrast the wild-type
mice injected with IL-2 had extensive to almost complete destruction of
the endothelial cell layer of the blood capillaries as well as
epithelial cell damage in the alveolar air spaces (Fig. 4a,
A2). The perforin KO mice injected with IL-2 had the least
amount of damage to the endothelial cells (Fig. 4b,
B2). Many endothelial cells appeared to be morphologically
normal, as observed in the controls. The IL-2-treated lpr
mice displayed more damage in the blood vessels, with shrinkage of
endothelial cells away from the basal lamina and extensive
deterioration of the endothelial and epithelial cells, which are
separated by the basal lamina (Fig. 4b, B3). The
most severe destruction of endothelial cells and epithelial cells of
the alveolar air spaces and breakage of the basal lamina occurred in
the IL-2-treated gld mice. In the lungs of these mice,
widespread 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.
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LAK activity in mice undergoing VLS
To test whether the lymphocytes from IL-2-treated mice would
exhibit increased LAK activity and to investigate the nature of
cytolytic effector molecules triggered by in vivo IL-2 administration,
splenic T cells collected from mice undergoing VLS were tested for
cytolytic activity against Fas+ YAC-1 tumor targets. The
data shown in Figure 5 (upper
panel) indicated that perforin-KO (B) and
gld (C) mice exhibited minimal spontaneous
cytotoxicity against YAC-1 targets. However, after IL-2 administration,
they exhibited a significant increase in cytotoxicity, although such
lytic activity was less than that seen in the wild-type mice
(A). These data indicated that IL-2 up-regulates both
FasL-based and perforin-based cytotoxicity, which may subsequently play
a role in endothelial cell lysis. To test the ability of IL-2-induced
LAK cells to kill endothelial cells, we used a well-characterized
endothelial cell line, TME-3H3 (13). The data shown in Figure 5
(lower panels) indicated that wild-type
(D), perforin-KO (E), and gld
(F) mice failed to exhibit spontaneous cytotoxicity of
endothelial cells. However, IL-2-induced LAK cells from wild-type mice
mediated the highest level of cytotoxicity, followed by those from
gld and perforin-KO mice. These data correlated well with
the VLS results (Fig. 1) in which perforin-KO mice were found to
exhibit marked decrease in VLS, and gld mice showed only a
partial decrease in certain organs. It should be noted that YAC-1 and
TME-3H3 cell lines expressed significant levels of Fas as determined by
flow cytometry (Fig. 6). However, the
endothelial cell line expressed lower levels of Fas, and only 42% of
the cells expressed Fas compared with the thymocytes from wild-type
mice, which expressed higher levels. This may explain why
endothelial cell lysis was more dependent on perforin than FasL as
shown in Figure 5 (lower panel).
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We and others have demonstrated that activation through CD44 can trigger lytic activity in CTL and NK/LAK cells (16, 17, 21, 22). Inasmuch as endothelial cells express the ligand for CD44, we have hypothesized that LAK cells expressing CD44 may spontaneously kill endothelial cells following interaction between LAK cells and endothelial cells (13, 17, 20). We therefore investigated whether IL-2 treatment would up-regulate CD44 expression in T cells. To this effect, wild-type, perforin-KO, gld, and lpr mice injected with IL-2 as described before and were sacrificed, and purified T cells from the spleens were stained using mAbs against CD44 and were analyzed flow cytometrically.
The data shown in Figure 7 indicated that
in IL-2-treated mice, there was significant up-regulation in CD44
expression on T cells in all groups of mice tested compared with that
in the controls. This was evident from the increase in the mean
intensity of fluorescence on T cells following IL-2 administration. It
should be noted that IL-2 treatment up-regulated the expression of
LFA-1 and L-selectin and down-regulated CD3 expression to a moderate
extent in all strains of mice (data not shown).
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To investigate the effects of IL-2 treatment on CD4+ T
and CD8+ T cells in the periphery and to compare the levels
of their induction in different groups of mice, the proportions of
CD4+ and CD8+ T cells in the spleens were
detected using flow cytometry. The data shown in Table II indicated that IL-2 treatment caused
an increase in the percentage of CD8+ T cells and a
consequent decrease in the percentage of CD4+ T cells in
the periphery of wild-type, perforin-KO, gld, and
lpr mice. These data indicated that IL-2 induced similar
activation of CD8+ in all strains of mice tested and that
the differences in cytotoxicity seen in various groups did not result
from altered activation of T cell subsets.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier studies have demonstrated that immunosuppression of mice by pretreatment with irradiation or injection of cyclophosphamide or cortisone markedly reduces or eliminates the development of IL-2-induced VLS (18). These data suggested that IL-2 does not act directly on the blood vessels to alter their permeability, but that it may do so indirectly by involving cells of the immune system. Vascular leaks in acute inflammatory lesions are triggered by mediators of immediate hypersensitivity, such as histamine, serotonin, and bradykinin (25). Although the IL-2-induced vascular leak resembles that triggered by the above-mentioned mediators, earlier studies have ruled out the involvement of vasoactive amines in IL-2-induced VLS (18). Lymphocytes are also known to produce a variety of mediators that increase the vascular permeability (26, 27). However, the current study demonstrates that IL-2-induced vascular leak may result from the involvement of cytolytic effector molecules, perforin and FasL.
Several studies have suggested that endothelial cells can be the targets of lymphocyte-mediated destruction. Damle et al. demonstrated that IL-2-activated LAK cells adhered and killed endothelial cells efficiently (10). Also, the toxicity associated with IL-2 therapy has been shown to decrease after depletion of NK cells in vivo (29). The role of CTL in VLS induction was also demonstrated in our earlier studies, in which it was noted that administration of a CTL clone plus IL-2 into irradiated syngeneic mice, but not the CTL clone or IL-2 alone, triggered VLS (13). Also, the IL-2-activated CTL clone could mediate efficient lysis of an endothelial cell line, but not a fibroblast cell line, in an MHC-unrestricted fashion (13). Moreover, methotrexate was found to attenuate pulmonary vascular leak by preventing the proliferation of NK/LAK cells and also by inhibiting leukocyte binding to endothelium (30). Similarly, dextran sulfate, which blocks leukocyte-endothelial adhesion, attenuates IL-2-induced VLS (31). Such studies together with the data presented in the current study suggest that IL-2-induced VLS may result from the direct cytotoxicity of endothelial cells by LAK cells.
In the current study it was observed that perforin-KO mice exhibited marked decrease in VLS in the lungs, whereas gld mice did not exhibit any decrease, but, in fact, exhibited a significant increase in VLS in the lungs. These data suggested that perforin played a key role in VLS induction in the lungs and that FasL was not critical. The fact that VLS in Fas-deficient (lpr) mice was of the same extent as that in the wild-type mice, further corroborated these results. In the current study it was not clear why the gld mice exhibited increased VLS in the lungs. This was seen in all three repeated experiments. It can be speculated that increased perivascular infiltration of lymphocytes as seen in the histopathology of lung tissue and/or increased susceptibility of lung endothelial cells to cytolytic activity may have triggered increased VLS in gld mice. Also, in gld mice, there was greater VLS in the lungs but less in the liver and spleen, thereby suggesting more damage to lung endothelium and less to the endothelium of spleen and liver. However, when LAK cells from gld mice were tested for cytotoxicity against the endothelial cell line in vitro, they exhibited similar levels of cytotoxicity as the wild-type mice. This may be because the endothelial cell line used was derived from the lymph node. Thus, it is possible based on our data that endothelial cells from various organs, following IL-2 treatment in vivo, may vary in their susceptibility to lysis by LAK cells. The fact that in the liver and the spleen, both the perforin-KO and gld mice had significantly diminished VLS suggested that both FasL and perforin may play a significant role in VLS induction in these organs. These data also suggested that the endothelial cells from different organs may exhibit differential susceptibility to perforin and FasL-mediated cytotoxicity. The fact that lpr mice also exhibited decreased VLS in the liver and spleen suggested that the endothelial cells in these organs may express Fas. The endothelial cell line used in the current study expressed significant levels of Fas. Whether the endothelial cells in different organs express varying levels of Fas, thereby accounting for the differential susceptibility to FasL-based cytotoxicity, remains to be determined. It should be noted that in the lungs, perforin, but not FasL, played an important role in VLS induction. However even in perforin-KO mice the VLS was not completely abolished. This suggested the possibility that other cytotoxic molecules such as TNF may play a role in VLS induction. It should be noted that the role of TNF in VLS is controversial. Studies involving attempts to block TNF have demonstrated either beneficial or no effect on IL-2-induced toxicity (32, 33). In addition, 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 of lymphocytes, but not neutrophils, in the perivascular tissue. These studies ruled out the possible damage to endothelial cells caused by neutrophils as reported in other models (36, 37, 38). The fact that IL-2-treated perforin-KO, gld, and lpr mice demonstrated significant perivascular infiltration of lymphocytes, in fact greater than that in the IL-2-treated wild-type mice, ruled out the possibility that the decreased VLS seen in the KO/mutant mice was because of the inability of cytolytic lymphocytes to migrate to the vascular tissues of various organs. Also, the fact that all strains tested exhibited similar up-regulation of CD44, a molecule involved in lymphocyte homing, further suggested that decreased VLS in the KO/mutant mice was due to the deficiency of the cytolytic effector molecules rather than lymphocyte migration and homing. Our data are consistent with previous histologic studies that showed lymphoid cell infiltration in lung, liver, kidney, and heart following IL-2 therapy (39, 40). Also, the electron microscopic studies confirmed that IL-2 treatment in wild-type mice caused significant damage to the endothelial cells, and furthermore, the results on VLS as seen in various mutant/KO mice correlated well with the ultrastructural studies on endothelial cell damage. It should be noted that the lpr and gld mice used in the current study were 4 to 6 wk of age, during which time they have similar proportions of T cells, as shown in our earlier studies on the thymus (41). This, however, did not rule out the possibility that such differences could exist in the periphery, particularly after IL-2 treatment. In the current study it was noted that IL-2 treatment caused a similar increase in the percentage of CD8+ T cells and a decrease in CD4+ T cells in all groups of mice. These data ruled out the possibility that the differences in the cytotoxicity and VLS induction resulted from differential activation of CD4+ or CD8+ T cells in various groups of mice.
Although in the current study we used IL-2-induced VLS as a model to study LAK cell-endothelial cell interactions, there is growing evidence that similar endothelial cell injury may occur in a variety of disease models. Thus, the vascular leak seen at sites of chronic inflammation involving mononuclear cells may be triggered by the direct killing of endothelial cells by cytolytic lymphocytes involving perforin and FasL-based pathways.
We and others have shown earlier that CTL, double-negative T cells and NK cells upon activation express high levels of CD44 and mediate efficient MHC-unrestricted TCR-independent lysis following ligation of CD44 (13, 17, 20, 21, 22, 42). We have also demonstrated that the lysis of endothelial cells ex vivo by cytolytic lymphocytes can be blocked by soluble CD44 fusion protein, anti-CD44 Fab, or soluble hyaluronate (unpublished observations). These data suggested that CD44-hyaluronate interactions may play an important role in the migration, homing, and lysis of endothelial cells. Further studies on the CD44 isoforms involved in lymphocyte adhesion and cytotoxicity of the endothelial cells should provide useful information on therapeutic intervention to prevent endothelial cell injury.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Prakash S. Nagarkatti, Department of Biology, Virginia Tech, Blacksburg, VA 24061. E-mail address:
3 Abbreviations used in this paper: VLS, vascular leak syndrome; KO, knockout; FasL, Fas ligand; PE, phycoerythrin.
Received for publication December 5, 1997. Accepted for publication May 11, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays a central role in interleukin-2-induced pulmonary vascular leak and lymphocyte accumulation. Cell. Immunol. 157:170.[Medline]
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 R. M. Licon Luna, E. Lee, A. Mullbacher, R. V. Blanden, R. Langman, and M. Lobigs Lack of both Fas Ligand and Perforin Protects from Flavivirus-Mediated Encephalitis in Mice J. Virol., March 7, 2002; 76(7): 3202 - 3211. [Abstract] [Full Text] [PDF]
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 V. ARNOLD, S. BALKOW, R. STAATS, H. MATTHYS, W. LUTTMANN, and J. C. VIRCHOW Jr. Increase in Perforin-positive Peripheral Blood Lymphocytes in Extrinsic and Intrinsic Asthma Am. J. Respir. Crit. Care Med., January 1, 2000; 161(1): 182 - 186. [Abstract] [Full Text]
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S. HASHIMOTO, A. KOBAYASHI, K. KOOGUCHI, Y. KITAMURA, H. ONODERA, and H. NAKAJIMA Upregulation of Two Death Pathways of Perforin/Granzyme and FasL/Fas in Septic Acute Respiratory Distress Syndrome Am. J. Respir. Crit. Care Med., January 1, 2000; 161(1): 237 - 243. [Abstract] [Full Text]
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 J. E. CARDIER, T. SCHULTE, H. KAMMER, J. KWAK, and M. CARDIER Fas (CD95, APO-1) antigen expression and function in murine liver endothelial cells: implications for the regulation of apoptosis in liver endothelial cells FASEB J, November 1, 1999; 13(14): 1950 - 1960. [Abstract] [Full Text]
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G. Matute-Bello, W. C. Liles, K. P. Steinberg, P. A. Kiener, S. Mongovin, E. Y. Chi, M. Jonas, and T. R. Martin Soluble Fas Ligand Induces Epithelial Cell Apoptosis in Humans with Acute Lung Injury (ARDS) J. Immunol., August 15, 1999; 163(4): 2217 - 2225. [Abstract] [Full Text] [PDF]
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A. Q. Rafi-Janajreh, D. Chen, R. Schmits, T. W. Mak, R. L. Grayson, D. P. Sponenberg, M. Nagarkatti, and P. S. Nagarkatti Evidence for the Involvement of CD44 in Endothelial Cell Injury and Induction of Vascular Leak Syndrome by IL-2 J. Immunol., August 1, 1999; 163(3): 1619 - 1627. [Abstract] [Full Text] [PDF]
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 M. Bradley, A. Zeytun, A. Rafi-Janajreh, P. S. Nagarkatti, and M. Nagarkatti Role of Spontaneous and Interleukin-2-Induced Natural Killer Cell Activity in the Cytotoxicity and Rejection of Fas+ and Fas- Tumor Cells Blood, December 1, 1998; 92(11): 4248 - 4255. [Abstract] [Full Text] [PDF]
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