The Role of Leukocytes Following Cerebral Ischemia: Pathogenic Variable or Bystander Reaction to Emerging Infarct?

Experimental Neurology 173, 168 –181 (2002) doi:10.1006/exnr.2001.7835, available online at http://www.idealibrary.com on

REVIEW The Role of Leukocytes Following Cerebral Ischemia: Pathogenic Variable or Bystander Reaction to Emerging Infarct? Dwaine F. Emerich,* Reginald L. Dean III,* and Raymond T. Bartus* ,† ,1 *Life Sciences Research and Development, Alkermes, Cambridge, Massachusetts 02139; and †Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts 02111 Received June 26, 2001; accepted October 3, 2001

Data accumulated over the last 10 years have led to the popular hypothesis that neutrophils and other inflammatory cells play a prominent role in the neuropathology of cerebral ischemia. This hypothesis was derived from a large number of studies involving three general observations: (1) leukocytes, particularly neutrophils, are present in ischemic tissue at the approximate time that substantial neuronal death occurs; (2) neutropenia is sometimes associated with reduced ischemic damage; and (3) treatments that prevent leukocyte vascular adhesion and extravasation into the brain parenchyma can be neuroprotective. This review reexamines the literature to ascertain its support for a pathogenic role for neutrophils in ischemia-induced neuronal loss. To accomplish this goal, we employed several logical theorems of “cause– effect” relationships, as they pertain to leukocytes and ischemic brain damage. Since the majority of studies focused on neutrophils as the most likely pathogenic inflammatory cell, this review necessarily does so here. We reasoned that if neutrophils play an important pathogenic (i.e., cause– effect) role in the neuronal damage that follows a stroke, then one should expect to find clear evidence that: (1) neutrophils invade the ischemic area prior to terminal stage infarction, (2) greater numbers of early appearing neutrophils are accompanied by evidence of greater neuronal loss, and (3) dose-related inhibition of neutrophil trafficking or activity produces a corresponding decrease in the degree of brain damage following ischemia. This review of the literature reveals that the existing evidence does not readily support any of these predictions and that, therefore, it consistently falls short of establishing a clear cause– effect relationship between leukocyte recruitment and the pathogenesis of ischemia. While the available evidence does not necessar1

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ily rule out a potential pathogenic role of neutrophils and other leukocytes, it nevertheless does expose serious weaknesses in the existing studies intended to support that hypothesis. For this reason we also offer suggestions for additional experiments and the inclusion of control groups that, in the future, might provide more effective or conclusive tests of the hypothesis. © 2002 Elsevier Science Key Words: neutrophil; neutrophil trafficking; ischemia; neutropenia; leukocytes; knockout animals; blood– brain barrier.

INTRODUCTION

Despite great advances in our understanding of the events that occur during and following brain ischemia, effective treatments for stroke are limited. Research over the past 2 decades has established that the majority of damage following a stroke does not occur immediately, but rather develops gradually over the course of many hours (6, 18, 76, 85). In fact, recent studies in animal models demonstrate that a surprisingly large number of intact and viable-looking neurons persist for several hours within the ischemic area (8). A large list of molecular, biochemical, and cellular changes have been identified as possible pathogenic variables in ischemic damage, providing optimism that pharmacological strategies will one day be developed to modulate the cascade of pathological events and thereby reduce ischemia-induced cell death. One of the hallmark responses to ischemia is a pronounced inflammatory response, which is characterized, in part, by the delayed recruitment of leukocytes (particularly neutrophils) into the ischemic zone. Given the wellestablished fact that neutrophils can induce local tissue damage and are among the earliest inflammatory cells to infiltrate an area, their possible contribution to the development of ischemic damage has clear intuitive appeal. Although the presence of neutrophils

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within infarcted tissue is consistently observed sometime after the ischemic event, their role in the pathogenesis of ischemia nonetheless remains uncertain. Proponents of the notion that neutrophils play a causative role in ischemic cell death suggest that neutrophils could contribute to the extent of ischemic damage by at least two different mechanisms. First, the aggregation and binding of neutrophils to endothelial cells could plug the blood vessels coursing through the ischemic region, thereby further restricting blood flow and exacerbating ischemic damage. Second, neutrophils could extravasate into the brain parenchyma and release cytotoxic substances (e.g., nitric oxide, elastase, and various cytokines) into the ischemic tissue, directly participating in the ongoing neuronal pathogenesis. Three lines of evidence have been put forth to support the role of neutrophil recruitment in ischemic cell death. These include (1) the presence of neutrophils within ischemic tissue at the approximate time that substantial infarction occurs, (2) the reduction of ischemic damage following neutropenic treatments, and (3) the observation that treatments which prevent neutrophil adhesion can be neuroprotective. While these data collectively provide circumstantial support consistent with a possible pathogenic role for neutrophils in stroke, they fail to provide conclusive evidence for such a role. The present review summarizes and critically evaluates the available data implicating a pathogenic role of neutrophil recruitment in ischemia, in light of alternative, viable explanations. Suggestions for further studies that might provide more definitive tests of the hypothesis are also provided. ESSENTIAL EVIDENCE FOR SUPPORTING A PATHOGENIC ROLE OF NEUTROPHILS IN STROKE

At the center of the hypothesis that neutrophils and other leukocytes are involved in ischemic cell death is the observation that neutrophils are frequently present within ischemic tissue. However, while the mere presence of neutrophils in ischemic tissue may be necessary, it is not sufficient to establish a pathogenic role in the formation of an infarct. For example, rather than exacerbating the damage, the recruitment of neutrophils may simply occur in response to the emergence of postischemic necrosis. In this scenario, the neutrophils could have a scavenging (and potentially useful) function within and around the infarcted tissue. In an effort to evaluate the strength of the evidence supporting a pathogenic role of neutrophils, the available published data are reviewed in the context of the following logical theorems associated with a cause– effect relationship. (1) Principle of temporal ordering. If neutrophils play an important role in the pathogenesis of postischemic neuronal death, then clear evidence should exist for significant neutrophil trafficking into the area of

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ischemia prior to the formation of the majority of the infarct. (2) Principle of dose escalation. If recruitment of neutrophils exacerbates cell death, then quantitative evidence of greater numbers of (early emerging) neutrophils should correlate with more extensive brain damage. (3) Principle of topographic relationship. If neutrophils participate in postischemic neuronal death, then brain regions exhibiting the greatest cell loss should also demonstrate the greatest evidence of (early emerging) neutrophil recruitment. (4) Principle of modulatory neuroprotection. Pharmacologic treatments that produce a dose-related inhibition of neutrophil trafficking or activity should produce a corresponding dose-related decrease in infarct volume following ischemia. The evidence pertaining to each of these suppositions is discussed below. THE PRESENCE OF NEUTROPHILS IN ISCHEMIC TISSUE

A large number of studies have evaluated infiltration of neutrophils and other inflammatory cells into ischemic tissue, using a wide range of measurements, including histology (4, 5, 21, 22, 31, 36, 47, 49, 53, 58, 63, 64, 79, 100), myeloperoxidase activity (1, 5, 10, 91), radioactivity following injections of radiolabeled neutrophils (2, 45, 70, 90), and SPECT imaging of neutrophils in humans (2, 90). While the majority of these studies document regions of cerebral ischemia with neutrophil infiltration, none satisfy any of the theorems described in the prior section, leaving it difficult to conclude what, if any, role neutrophils play in ischemic brain damage (see Tables 1 and 2). A more detailed discussion of these studies helps illustrate this point. Certain studies failed to provide either a systematic and independent confirmation of neutrophil infiltration or any quantification of infarct size (4, 49, 75). Logically, of course, without both variables measured and compared, it is impossible to draw any cause– effect relationship (notwithstanding the authors’ claims to the contrary). An even more common interpretive problem is the universal lack of a demonstration that neutrophils invade the ischemic tissue prior to the formation of the end stage infarct (1, 2, 4, 5, 10, 21, 22, 30, 31, 35, 36, 39, 45, 47, 49, 53, 58, 59, 63, 64, 70, 75, 79, 90, 91, 100). Indeed, few studies even attempted to establish a temporal relationship, thus failing to offer the minimal evidence required to entertain causal possibilities. Rather, neutrophils or other leukocytes are typically reported within the ischemic tissue only after the vast majority of the infarct has developed, therefore providing no insight regarding

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TABLE 1 Rodent Studies Testing for the Presence of Neutrophils in Ischemic Tissue Do Not Support a Pathogenic Role of Neutrophils Study

Earliest increase in neutrophils (hours)

Site of increased neutrophils

24* 48* 72 12 6 0.5 12 12 12* 24 48* 24* 12* 6* 24 12* 21* 21* 24 24 24 0.25–1*

Vasculature and parenchyma Primarily parenchyma Parenchyma Primarily vascular Vasculature and parenchyma Vasculature Primarily parenchyma Parenchyma Parenchyma Parenchyma Parenchyma Parenchyma Parenchyma Parenchyma Parenchyma Vasculature and parenchyma Vasculature and parenchyma Vasculature and parenchyma Parenchyma Vasculature and parenchyma Vasculature and parenchyma Vasculature

Barone et al. (5) Dereski et al. (31) Garcia et al. (39) Clark et al. (21) Clark et al. (22) Garcia et al. (36) Matsuo et al. (64) Matsuo et al. (63) Schroeter et al. (79) Zhang et al. (100) Barone et al. (4) Jander et al. (49) Yamasaki et al. (91) Hayward et al. (47) Kato et al. (53) Lehrmann et al. (58) Ahmed et al. (1) Ritter et al. (75)

Earliest ischemic damage (hours)

Not Not Not

Not

24* a 12 72 2–6 1–6 6–12 6–12 24 a 24 a 24 a 6* 6* determined determined determined 168 a 12* 4* 4 6 24 a determined

Temporal priority for neutrophils Temporal relationship not tested Neutrophils appeared after infarct Neutrophils appeared after infarct Neutrophils appeared after infarct Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Neutrophils appeared after infarct Neutrophils appeared after infarct Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Neutrophils appeared after infarct Neutrophils appeared after infarct Neutrophils appeared after infarct Neutrophils appeared after infarct Temporal relationship not tested Temporal relationship not tested

Note. While neutrophils are consistently observed within the area of cerebral infarction, their contribution to the development of the infarct remains uncertain. The hypothesis that neutrophils play an important pathogenic (i.e., cause– effect) role in focal ischemia requires evidence that neutrophils invade the ischemic area in advance of the major brain damage. As shown above and elaborated in the text, no studies have satisfied this essential criterion. *Statistically significant (in all other studies statistics were not reported). a Only “end stage” infarct time was assessed.

temporal ordering necessary for suggesting a causal relationship. The few notable exceptions that did attempt to characterize the temporal relationship between inflammatory cell accumulation and infarct formation deserve special discussion. Ironically, while most of these studies concluded a probable pathogenic role of neutrophils, a close examination of the data derived from these

studies does not support that conclusion. For example, Dereski and colleagues (31) examined neutrophil accumulation at multiple time points and reported that significant accumulation did not occur until 48 h following the initiation of ischemia. While no morphometric determinations of infarct volume were provided in these studies, the neutrophil accumulation at 48 h was easily outside the time frame of when many other

TABLE 2 Nonrodent Studies Testing for the Presence of Neutrophils in Ischemic Tissue Do Not Support a Pathogenic Role of Neutrophils Study (species) Garcia and Kamliyo (35) (squirrel monkey) Hallenbeck et al. (45) (dog) Bednar et al. (10) (rabbit) del Zoppo et al. (30) (baboon) Pozzilli et al. (70) (human) Wang et al. (90) (human) Akopov et al. (2) (human) Lindsberg et al. (59) (human)

Earliest increase in neutrophils

18 hours 1–4 hours* 7 hours 1 hour 48 hours 7 days* 6–12 hours* 15 hours

Site of increased neutrophils

Earliest ischemic damage (hours)

Temporal priority for neutrophils

Parenchyma Parenchyma Vasculature and parenchyma Vasculature Vasculature and parenchyma Parenchyma Vasculature and parenchyma Vasculature

12 Not determined Not determined Not determined 48 Not determined 6–12 15

Neutrophils appeared after infarct Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested Uncertain (see text for discussion) Temporal relationship not tested

Note. Similar to the rodent studies listed in Table 1, studies using other species have also failed to establish that neutrophils appear in advance of major infarct. Thus, they also fail to support the hypothesis that neutrophils play an important pathogenic role in focal ischemia. *Statistically significant (in all other studies statistics were not reported).

LEUKOCYTES AND CEREBRAL ISCHEMIA

studies have demonstrated that peak damage occurs in this model (e.g., approximately 12 h postocclusion). Even though these data were interpreted as support for a role of neutrophils in ischemic damage, the experimental design did not actually allow any definitive interpretation to be made. More to the point, however, a more parsimonious explanation of the data would seem to be that the neutrophil infiltration that did occur did not contribute to, but was merely the result of, the cerebral infarct. Zhang et al. (100) measured neutrophil accumulation and infarct formation between 6 and 168 h after both permanent and transient occlusions of the MCA in rats. Small numbers of neutrophils were observed within the parenchyma of the ischemic tissue between 6 and 12 h after occlusion. Importantly, they also observed that significant cell death had already occurred by this time. Moreover, they reported that near maximal cell damage was seen by 12 h, a time when neutrophil accumulation was still relatively low. Indeed, maximum neutrophil accumulation did not occur until much later, between 24 and 48 h. Importantly, infarct volume did not appreciably change as a result of this accumulation (i.e., peak volume occurred at 12 h). These data, therefore, also appear to be more consistent with the idea that the neutrophils were accumulating in response to the formation of an infarct, rather than as an important precipitating or contributing event. Another interesting point of this study was that when the temporal data from the two models (i.e., permanent and transient occlusion) were compared, the rate of neutrophil accumulation and infarct formation were negatively, not positively, correlated. That is, the condition precipitating the greatest numbers of parenchymal neutrophils produced the smallest infarct volume. Of course, this observation is also at odds with the “pathogenic neutrophil hypothesis.” Hayward et al. (47) similarly conducted a detailed series of studies using two different rat models of focal ischemia (i.e., partial and complete reperfusion). These studies also suggested that the accumulation of neutrophils does not exacerbate the volume of the infarct, but rather occurs as a more delayed reaction to the infarct’s formation. As in the prior Zhang et al. study (100) the accumulation of neutrophils into the infarcted region consistently occurred after the development of the infarct. For instance, following focal ischemia with partial reperfusion, neutrophil accumulation was not observed until 21 h after surgery, even though the infarct was completely developed at approximately 12 h. Similar results were obtained with the complete reperfusion model. In fact, very few neutrophils were seen prior to the time that maximal damage was observed, clearly indicating that neutrophil accumulation did not contribute to the development of the infarct.

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Finally, despite numerous methodological and interpretive ambiguities, the intriguing data from one human study that attempted to track neutrophils following strokes deserves special discussion (2). Autologous neutrophils labeled with technetium-99m were used with SPECT to monitor changes in neutrophil accumulation over time (from 3 to 6 h, postischemia to 30 days). As in prior studies in both animals and humans, only “minimal” neutrophil accumulation was observed in the ischemic tissue within the initial 6 h. A modest increase was then seen over the next 6 h and peak levels occurred between 12 and 24 h. The authors then employed a post-hoc analysis of the 12-h neutrophil data to define three different subgroups (based on the extent of neutrophil accumulation). They reported that the subgroup with the greatest neutrophil accumulation (at 12 h) exhibited the largest infarct volume (at 6 to 9 days) and most persistent neurological impairments. Clearly, these data are sufficiently intriguing to merit replication. Moreover, replication is essential prior to drawing firm conclusions because of a number of issues with the existing study. These include (1) the only new data presented were derived from the posthoc analysis; all other aspects of the accumulation of neutrophils are consistent with the prior, inconclusive studies reviewed here, (2) for unexplained reasons, this same post-hoc analysis excluded any patient who had a Mathew Scale (i.e., neurological status) of ⬎80 (i.e., mild deficit); it would have been insightful to determine the extent to which neutrophils accumulated in these relatively mild patients, for they would have served as important negative controls; (3) despite the authors’ arguments that all three subgroups were initially very similar (and that the differences in outcome only emerged later, after differences in neutrophils were manifest), the group with the greatest damage and neutrophil accumulation had shown trends of increased infarct volume, decreased neurological scores, and increased neutrophil accumulation at the earliest time points presented; the possibility is therefore raised that subtle but important differences may have existed in the subgroups from the onset and that the groups were not equivalent, as assumed. In conclusion, despite numerous studies that redundantly confirm the appearance of leukocytes within infarcted brain tissue, none provide clear evidence that the accumulation participates in, worsens, or accelerates the formation of the infarct. Of course, once a significant volume of infarcted brain tissue develops, a number of secondary pathologic and inflammatory events are likely to be induced, including a breakdown in the blood– brain barrier (reviewed in Dietrich (32)). It should not, therefore, be surprising that in time, significant accumulation of neutrophils and other reactive cells will be detected within the damaged region. However, the timing of their detection does not support

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TABLE 3 Neutropenia Treatment Studies Fail to Establish a Clear Link between Neutropenia and Infarct Reduction Studies

Preinfarct reduction of neutrophils? (%)

Treatment

Dutka et al. (33)

Mechlorethamine

95*

Bednar et al. (11) Helps and Gorman (48)

Antineutrophil serum Mechlorethamine

84* 70*

Shiga et al. (82)

Antineutrophil antibody Antineutrophil Antineutrophil Antineutrophil antibody Antineutrophil

78*

Chen et al. (16) Lopez and Lanthorn (61) Matsuo et al. (63) Connolly et al. (26) Hayward et al. (47) Kitagawa et al. (54)

Shimakura et al. (83)

monoclonal serum serum monoclonal antibody

Cyclophosphamide Antineutrophil antibody used to induce neutropenia in I-CAM knockout mice Antineutrophil antibody

Not determined No change 93* Not quantified; “near complete agranulocytosis” reported ⬎98* Not quantified; agranulocytosis reported

92*

Percentage of reduction in infarct size (or improved function) Attenuated decline in evoked response and cerebral blood flow* 46* Prevented decline in evoked response and cerebral blood flow Not determined 43* 40* ⬃60* 66* No reduction 36 compared to knockouts without neutropenia*

50*

Note. Massive depletions in neutrophils have often (though not universally) decreased infarct size or improved function following focal ischemia. However, a clear relationship between neutropenia and neuroprotection remains uncertain for several reasons. First, some of these neutropenia studies warn that neutropenia, per se, may not be responsible for the reduction in infarct (1, 61, 83). This possibility, coupled with the potentially severe and diverse physiological effects associated with the neutropenic manipulations, severely compromises any simple interpretation of these data. Moreover, several conventional controls have yet to be employed (see text) that are essential for establishing that the changes in infarct size are dependent upon changes in neutrophil numbers and not due to other confounding possibilities. *Statistically significant (in all other studies statistics were either not significant or not reported).

a pathogenic role of neutrophils in ischemic brain damage. ANTI-NEUTROPHIL INTERVENTIONS IN ISCHEMIA

A second experimental paradigm takes advantage of the advances in leukocyte biology responsible for new compounds to induce neutropenia, impede neutrophil trafficking, and inhibit bioactive secretions of activated neutrophils. All of these studies attempt to pharmacologically reduce the impact of neutrophils at the ischemic site to determine whether this also reduces the extent of ischemic brain damage. Effects of Neutropenia on Ischemia Several studies have examined the effects of various treatments intended to deplete levels of circulating neutrophils (11, 16, 26, 33, 47, 48, 54, 61, 63, 82, 83). On the surface, many (but not all) of these experiments appear to suggest some role of neutrophils in the development of cerebral infarction following ischemia, for massive depletions of neutrophils (typically ⬎90%) prior to ischemia have been reported to correlate with decreases in infarct size (Table 3). For instance, Chen et al. (16) reported that administration of antirat neutrophil serum to rats reduced infarct volume by a modest 30% (although no independent confirmation of neu-

tropenia was provided). Matsuo et al. (63) also reported that treatment with the antineutrophil monoclonal antibody RP3 reduced myeloperoxidase (MPO) activity (a marker of neutrophil accumulation) by 90%, which correlated with a 60% reduction in infarct size. Although these studies appear to support a role of neutrophil trafficking in cell death, a closer examination of all the available data reveals several interpretive inconsistencies and problems. For instance, Hayward et al. (47) used cyclophosphamide to induce nearly complete neutropenia, but found no effect on infarct volume. Even more intriguing are the results of Lopez and Lanthorn (61), who reported that while the antineutrophil serum used by Chen et al. (16) above significantly decreased the size of an MCA-occlusion infarct, no decrease in levels of plasma neutrophils was produced (i.e., neutropenia was not produced by the antineutrophil serum, emphasizing the deficiency mentioned in Chen et al.’s experimental design, above). The authors appropriately suggested these data thereby raise the distinct possibility that some mechanism other than inhibition of neutropenia trafficking could be responsible for the beneficial effects they and others observed with manipulations intended to induce neutropenia. More recently, Shimakura et al. (83) used a polyclonal antineutrophil antibody to induce neutropenia (which was 90% successful, as measured in the

LEUKOCYTES AND CEREBRAL ISCHEMIA

serum). They reported data reminiscent of those of Lopez and Lanthorn (61), for a modest (e.g., about 33%) decrease in infarct volume was seen, but with no decrease in neutrophil accumulation observed within the infarcted tissue. Thus, these data compromise attempts to form a simple relationship between a reduction in neutrophils and reduction in ischemic damage and add further credence to Lopez and Lanthorn’s suggestion that some uncontrolled (and currently undefined, nonneutrophil) variable may contribute to the reduced infarct reported. This point is further emphasized by a laudable study that attempted to increase (rather than decrease) leukocytes, assuming that if they were important modulators of ischemic cell death, then an even larger infarct would be induced after ischemia (1). Intravenous administration of lipopolysaccharide 24 h prior to ischemia indeed produced a marked increase in neutrophil infiltration into the ischemic region, as intended. Interestingly, despite significantly increasing the numbers of circulating leukocytes, a significant decrease in infarct volume was observed following cerebral ischemia. Clearly, this observation is directly contradictory to any simple cause– effect relationship between leukocytes and ischemic damage and raises more questions than it answers. The data provided in these latter studies (1, 61, 83) are particularly important, not simply because they raise questions regarding the conventional interpretation of the neutrophil trafficking experiments, but also because they offered the opportunity to directly link changes in neutrophil activity with ischemic outcome. Importantly, in each case, the data failed to support a cause– effect relationship. Finally, aside from these limitations with the neutropenia data which address the leukocyte hypothesis, a note of caution should be made regarding all of the neutropenia studies. Neutropenia involves a relatively drastic pharmacological manipulation, with potentially profound effects on the physiology and hemodynamics of the subject (46). These confounding effects make any simple interpretation of the neutropenia manipulations risky. Conventionally, in studies using drugs with diverse or complex effects, dose–response information is collected to reduce the interpretive risk. In the present instance, it would be important—perhaps essential—to determine whether a range of neutropenic doses produces a similarly graded decrease in both neutrophils and infarct volume. Unfortunately, no one has yet attempted to generate this minimal, conventional evidence. Additionally, another useful control that has not been run would involve replacing neutrophils in neutropenic rats to determine whether the infarct in this group would be restored to its normal (i.e., nonneutropenic size). Such an outcome would more clearly establish a link between neutrophils and ischemic damage, independently of some of the confounding changes that are induced by neutropenic

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drugs. Since these important control groups have not been incorporated into past experimental designs, valid questions persist regarding the interpretation of the mixed results. Moreover, even when positive effects are reported, the decreases in infarct size are relatively small (Table 3) compared to the 75–90% reductions often obtained using other pharmacological treatments, including glutamate antagonists and calpain inhibitors (7, 9, 41). For all these reasons, these data fall short of providing definitive support for a cause– effect relationship. Effects of Altering Cell Trafficking on Ischemia Other studies have used more sophisticated and presumably more selective means of altering neutrophil trafficking. These have included anti-ICAM-1 (14, 19, 64, 81, 96, 97) and anti-CD11/CD18 antibodies (13, 17, 19, 20, 23, 24, 37, 52, 64, 65, 99) as well as numerous other approaches (3, 12, 28, 38, 43, 44, 50, 51, 56, 60, 62, 66, 67, 69, 71, 73, 74, 87– 89, 92–95, 98). A review of these studies reveals that they also have failed to satisfy the basic theorems required to support a pathogenic role of neutrophils in ischemia (Table 4). While reductions in infarct size (19, 63, 81, 96, 98) and improvements in neurological outcome (13, 14, 56) have been reported, other studies have failed to find any evidence of reductions in lesion size following similar treatments (37, 44, 50, 52, 88, 96). Still other studies using neurological outcome as a marker of the extent of ischemic damage failed to find any positive benefit from preventing neutrophil trafficking. For instance, Takeshima et al. (88) were unable to demonstrate any difference in recovery of somatosensory evoked potentials or lesion volume in cats treated with a monoclonal leukocyte antibody. Similarly, Clark et al. (23, 24) failed to find any improvements in neurological outcome in rabbits following treatment with an anti-CD18 antibody. What is particularly notable about the cell trafficking studies (Table 4) is the virtual lack of an independent determination of neutrophil levels or activity. Rather, these studies consistently assume that the treatment produced the desired effect on neutrophils, but offer no confirmatory data. Accordingly, these data fail to adequately test for a cause– effect relationship. Thus, even those studies that report a decrease in infarct volume fall short of supporting a clear pathogenic role of neutrophils. Studies Using Gene Knockout Animals An alternative to pharmacologically manipulating neutrophils takes advantage of recent advances in molecular biology that provide knockout animals deficient in specific genes with a purported role in neutrophil activity and/or trafficking. Animals deficient in either the CD18 (72) or ICAM-1 gene (26, 54, 86) have demonstrated significant reductions in ischemic infarct

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TABLE 4 Antileukocyte Treatment Studies Fail to Establish a Clear Link between Neutrophil Trafficking/Activity and Infarct Reduction

Treatment approach

Studies

Treatment

Percentage of reduction in infarct size (or improved function)

Percentage of preinfarct reduction of neutrophils?

Relationship between the degree of neutrophil trafficking and ischemic damage?

⬃66* (Improved neurological score)* ⬃75* 41* No reduction 54* 42*

30* Not determined

No No

Not Not Not Not Not

determined determined determined determined determined

No No No No No

(Neurological score not improved) (Increase in vascular reflow)* 47* 28* No reduction 38* 43* 61* No reduction 32* (Improved neurological score)* 28

Not determined

No

Not determined

No

Not Not Not Not Not Not Not Not Not

determined determined determined determined determined determined determined determined determined

No No No No No No No No No

Not determined

No

Not Not Not Not Not

determined determined determined determined determined

No No No No No

Anti-ICAM-1 Shiga et al. (81) Bowes et al. (14)

Cyclosporin A ␣-ICAM-1 antibody

Matsuo et al. (64) Zhang et al. (97) Zhang et al. (96) Chopp et al. (19)

1A29 1A29 1A29 1A29 1A29

Clark et al. (23, 24)

Anti-CD18

Mori et al. (65)

Anti-CD18 (IB4)

Chen et al. (17) Chopp et al. (20) Jiang et al. (52)

Garcia et al. (37) Chopp et al. (19) Bowes et al. (13)

Anti-CD11b (1B6c) Anti-CD11b (1B6c) Anti-CD11b (1B6c) Anti-CD11b (1B6c) Anti-CD11a (WT1) Anti-CD18 (WT3) Anti-CD11b/18 Anti-CD11b Anti-CD18

Zhang et al. (99)

Anti-CD18

Dawson et al. (28) Barone et al. (3)

TNF binding protein Anti-TNF Mab Soluble TNF receptor TNF binding protein TNF binding protein

Anti-CD11/CD18

Matsuo et al. (64)

Anti-TNF

Nawashiro et al. (67) Nawashiro et al. (66)

32–41* 20* 26* 25* 26*

IL-1 receptor antagonist (ra) Relton and Rothwell (74) Garcia et al. (38)

IL-1ra

50*

Not determined

No

IL-1ra

Loddick and Rothwell (60) Relton et al. (73)

IL-1ra

41* 64* 72*

Not determined Not determined No

No No No

IL-1ra

45–46*

Not determined

No

48*

Not determined

No

No reduction

Not determined

No

58* 30–51*

Not determined Not determined

No No

Not determined Not determined

No No

Not determined Not determined No

No No No

Neutrophil inhibitory factor Jiang et al. (51) Jiang et al. (50)

Neutrophil inhibitory factor Neutrophil inhibitory factor

PAF Antagonist (Platelet Activating Factor) Bielenberg et al. (12) Kochanek et al. (56)

Apafant Kadsurenone

Gross et al. (43) Prehn et al. (71) Gross et al. (44)

TGF-␤1 TGF-␤1 TGF-␤1

47–59* (Recovery of evoked response)*

TGF-␤1 (transforming growth factor) 48* 10* No reduction

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LEUKOCYTES AND CEREBRAL ISCHEMIA

TABLE 4—Continued

Treatment approach

Studies

Treatment

Takeshima et al. (88)

MoAb60.3 (monoclonal leukocyte antibody) Hypothermia Heparin (antiadhesion) Dextran sulfate F20 peptide (antiadhesion peptide) Anti-IL-8 (WS-4) (mouse monoclonal antibody) TG-1 (antiadhesion peptide) Fibronectin peptide V (antiadhesion peptide) IL-10 Proteasome inhibitor (PS519)

Percentage of reduction in infarct size (or improved function)

Percentage of preinfarct reduction of neutrophils?

Relationship between the degree of neutrophil trafficking and ischemic damage?

No reduction

Not determined

No

Not Not Not Not

No No No No

Other targets

Toyoda et al. (89) Yanaka et al. (94, 95) Zhang et al. (98)

Matsumoto et al. (62)

Yanaka et al. (93) Yanaka et al. (92)

Spera et al. (87) Phillips et al. (69)

Shimakura et al. (83)

59* 41–60* 61* 40*

ONO-5064 (neutrophil elastase inhibitor)

determined determined determined determined

60*

No

No

33*

Not determined

No

50*

Not determined

No

31–40* ⬃60*

Not determined Not determined

No No

⬃40* ⬃28*

Not determined Not determined

No No

Note. While modest reductions in infarct size have been observed following a variety of antileukocyte treatments, these studies have not fulfilled several logical criteria required to conclude that inhibiting cell trafficking, per se, is responsible for the reduction in infarct volume. To establish a cause– effect relationship for inhibition of cell trafficking and a reduction in infarct volume, minimally one must observe (1) a reduction in cell trafficking prior to the development of ischemic brain damage and (2) a relationship between the extent to which cell trafficking is inhibited and the reduction of ischemic damage (i.e., conventional dose–response requirement). Neither of these criteria have been met, thus falling short of establishing a cause– effect relationship. *Statistically significant (in all other studies statistics were either not significant or not reported).

size, relative to that of wild-type animals (Table 5). One notable paper also reported improvements in neurological outcome, decreased mortality, and improved

cerebral blood flow within the ischemic penumbra (26). However, none of these studies confirmed that neutrophil tracking was improved in the knockout animals

TABLE 5 Studies with Knockout Mice Do Not Establish a Link between Neutrophil Trafficking/Activity and Infarct Reduction Studies Prestigiacomo et al. (72) Connolly et al. (26) Soriano et al. (86) Kitagawa et al. (54) Bruce et al. (15) Gary et al. (40)

Schielke et al. (77) Grilli et al. (42)

Knockout gene CD18 CD18 ICAM-1 ICAM-1 ICAM-1 p55/p75 TNF receptor p55 TNF receptor p75 TNF receptor p55/p75 TNF receptor IL-1␤ converting enzyme IL-10

“Knockout” reduced infarct size No difference 53% decrease* 73% decrease* 82% decrease* 87% decrease* 33–83%* 27% increase* 24% increase* No difference 20% increase* 52% decrease* 30% increase*

Preinfarct reduction of neutrophils? Temporal relationship not tested Temporal relationship not tested No quantification of neutrophils Temporal relationship not tested Temporal relationship not tested Temporal relationship not tested No quantification of neutrophils No quantification of neutrophils No quantification of neutrophils No quantification of neutrophils No quantification of neutrophils No quantification of neutrophils

Note. Knockout animals lacking specific genes with a suspected role in neutrophil trafficking and/or activity generally show significant decreases in infarct size, relative to wild-type animals. However, no study has yet confirmed that genetic manipulation reduces the extent of neutrophil invasion into the ischemic region, especially during the early pathogenic period. Accordingly, none of these studies has provided clear support for a cause– effect relationship between neutrophils and ischemic damage. *Statistically significant.

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TABLE 6 Antineutrophil Trafficking: Clinical Trials Have yet to Demonstrate Efficacy Study

Treatment (sponsor)

Mechanism

Clinical phase (No. of patients)

Treatment initiated (time)

EAST (Enlimomab Acute Stroke Trial) (34)

Enlimomab (Boerhinger Ingelheim)

ICAM-1 monoclonal antibody

Phase II (625)

⬍6 h after onset symptoms

HALT (Hu23F2G Anti-adhesion to Limit cyto-Toxic injury) (80)

Hu23F2G LeukArrest (ICOS)

CD11/CD18 monoclonal antibody

Phase III (310)

Unknown

Neutrophil inhibitory factor (27)

UK-279,276 (Corvas and Pfizer)

CD11b/CD18 antagonist

Phase IIb (ongoing)

Unknown

Status Terminated: Neurological outcome, measures of adverse events (fever and infections), and mortality increased in treatment group. Terminated: interim results indicated drug did not show any clinical benefit as determined by neurological outcome. Ongoing

Note. Clinical trials conducted to date examining the functional outcome of inhibiting neutrophil trafficking in stroke patients. Of the three trials publicly reported, two have been terminated due to lack of patient benefit and/or unacceptably high rates of adverse treatment events. A third trial (Phase IIb) is ongoing.

prior to infarct formation and that changes in neutrophil tracking were therefore responsible for the reduction in infarct volume. Additionally, not all results have been as positive as these. For instance, Prestigiacomo and colleagues (72) found that while CD18 knockouts subjected to transient ischemia showed significant reductions in infarct (53%), relative to wild-type animals, no improvements in neurological outcome were found in these animals. Moreover, the reductions in infarct size were not apparent when the model was altered to include permanent, rather than transient, ischemia. In a second study, Kitagawa et al. (54) found that ICAM-I knockouts that received permanent occlusion of the MCA showed a modest, 33% reduction in infarct size, compared to that of wild-type animals. Despite these neuroprotective effects of knocking out the ICAM-1 gene, no differences in the numbers of MPO-positive cells in the cortex and striatum between the knockout animals and controls (wild-type) were seen. This unexpected observation raises the logical question of exactly what other (perhaps compensatory) changes may exist in the knockout animals that might influence infarct volume and what role, if any, neutrophils (i.e., MPO-positive cells) play in the observed effect. More to this point, because many of the knockout studies have not attempted to quantitate neutrophils and those that did have not monitored the infiltration of neutrophils into the ischemic tissue over time, they fail to establish the necessary linkage between neutrophil trafficking and the extent of ischemic damage. While these data collectively have been interpreted as support for a role of neutrophils in ischemic damage, the experimental designs do not allow cause– effect interpretations to be made.

ANTINEUTROPHIL TRAFFICKING: CLINICAL TRIALS IN STROKE PATIENTS

To date, three compounds, whose primary mechanism of action is purported to involve inhibition of neutrophil trafficking, have been evaluated in separate clinical trials. Of these three trials, two have been terminated due to poor patient outcome, while the third is ongoing (Table 6). The first study, EAST (the Enlimomab Acute Stroke Trial) was a 62-center, Phase III trial involving a total of 625 patients. Patients were treated with a mouse monoclonal antibody to ICAM-1 (Enlimomab) or placebo for 5 days (beginning within 6 h from the onset of stroke symptoms). Overall, treated patients actually exhibited a worse functional outcome, compared to that of placebo controls (34). They also displayed greater adverse events (fever, 51% vs 27%; infections, 55% vs 42%) and a higher mortality rate (22% vs 16%). While the reason for disappointing treatment outcomes and adverse treatment effects is unknown, one possibility is that the treated patients developed an immune response to the mouse antibody (25, 29). Data supporting this possibility come from the human antimurine antibody response to Enlimomab reported in an openlabel clinical trial with 32 stroke patients. Of the 27 evaluable patients, 100% had detectable IgG responses and 93% had IgM responses to Enlimomab (78). Another Phase III trial (HALT, Hu23F2G anti-Adhesion to Limit cyto-Toxic injury) used a humanized antibody to CD11/CD18 (LeukArrest; Hu23F2G). Three hundred and ten patients were treated for 3 days using a previously determined maximum tolerable dose of 1.5 mg/kg given twice a day (84). While no adverse effects were observed, an interim analysis of

LEUKOCYTES AND CEREBRAL ISCHEMIA

the data failed to find any significant clinical benefit. For this reason, the trial was terminated. These data are similar to the other LeukArrest Phase II clinical trials for treatment of hemorrhagic shock, multiple sclerosis, and cardiac ischemia, which also failed to find any benefit from inhibiting or blocking CD11/ CD18 (80). Finally, a non-antibody CD11b/CD18 antagonist (recombinant neutrophil inhibitory factor (rNIF); UK-279,276; originally isolated from the blood-feeding hookworm) is currently being tested in a Phase IIb trial in stroke patients to determine its efficacy using functional and neurological outcomes over a wide dose range (27). It remains too early to know whether this unique approach will be any more successful than the two that used the antibody approach. In summary, the lack of positive clinical findings may not be entirely surprising, given that the review of current preclinical studies does not yet provide convincing overall support for the neutrophil hypothesis. However, several practical factors may explain the lack of positive effects, including testing too narrow a dose range, administering the treatment too late following initiation of the pathogenic cascade, and missing a possible positive effect by measuring the end points at a suboptimal time point. Nonetheless, the lack of detailed preclinical information regarding the timing and magnitude of a possible response, relative to the development of ischemic damage (as critiqued here) deprives clinicians of the type of information essential for designing clinical protocols with a high probability of success. ADDITIONAL PERSPECTIVE ON ANIMAL MODELS

Throughout this review we have offered a logical framework and specific suggestions for experimental designs that might provide more effective and valid tests of the “neutrophil hypothesis.” However, it should also be noted that even if more effective tests of the hypothesis are conducted, the animal models typically employed might continue to complicate predictions of clinical outcomes. First, it has been suggested, generally, that animal models of ischemic brain injury (typically involving young, healthy animals) may be too forgiving, thus producing many false positive drug outcomes not likely to be confirmed in the clinic (6). Second, it has been argued that the animal species used in the preclinical models for ischemia and inflammation may not adequately mimic the biochemical and immunological complexities of the human inflammatory response pathway (29), complicating the leap from animal experiments to clinical applications. Finally, a close examination of the types of models employed in this field raises yet another concern. To date, the vast majority of studies attempting to demonstrate a relationship between neutrophils and neuroprotection

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have used only two models: the intraluminal suture model or artificial embolism models. Electron microscopy studies have demonstrated that the suture method causes significant damage to the vascular tissue, particularly the endothelial cells (68). This physical trauma, which includes severe denudation of portions of the luminal endothelium, additional injury to the smooth muscle layer, and damage to the tight junctions comprising the blood– brain barrier, is independent of cerebral ischemia and artifactual to the model’s methodology. Of course, reliance on a model that damages the vasculature in this manner is likely to artificially increase the neutrophil response beyond that induced by the ischemic episode. Similarly, embolism models have been reported to cause extensive injury to endothelial cells and other constituents of the blood– brain barrier (55, 57). Thus, both popular models for the tests of the neutrophil hypothesis likely exaggerate recruitment of neutrophils to the vasculature near the ischemic brain tissue, offering a view of neutrophil involvement that may exceed rather than be representative of the clinical setting. For this reason, the use of alternative means of vessel occlusion to induce ischemia would help resolve future interpretive problems likely to arise, as outlined here. SUMMARY AND CONCLUSIONS

An extensive literature has developed over the past 10 years that is commonly interpreted as supporting the hypothesis that neutrophils and other leukocytes play a prominent role in the neuropathology of ischemia. In this review we have attempted to objectively assess the published data, in view of logical requirements for establishing a cause– effect relationship. Despite a number of studies that superficially provide circumstantial support, the bulk of the evidence accumulated to date fails to provide strong support for the hypothesis. Indeed, a number of studies offer data that contradict a pathogenic role of leukocytes. To help provide logical structure to this review of the literature, we posited several theorems consistent with the hypothesized cause– effect relationship. This review revealed that the vast majority of efforts have thus far failed to satisfy these essential tenets, including the need to demonstrate that enhanced leukocyte trafficking or activity clearly precedes ischemia-induced cell death (Tables 1 and 2), that a relationship exists between the degree of early neutrophil trafficking and the degree of brain damage (Tables 1 and 2), and that the neuroprotection produced by antileukocyte treatments is clearly associated with changes in neutrophil activity or levels (Tables 3 and 4). Thus, although the hypothesis that neutrophils contribute to the extent of ischemic damage enjoys a certain intuitive appeal, the empirical evidence generated to support the hypothesis remains inconclusive, at

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best. The overriding problem revealed by this review is that the vast majority of studies seemed poorly designed to offer genuine tests of the hypothesis, but rather were better designed to generate additional, circumstantial (and inconclusive) support. We have therefore attempted to offer explicit suggestions for additional control groups and empirical approaches that may help future studies provide a more definite test of the role of leukocytes in ischemia. Until the problems identified in this review are addressed and corrected, the pathogenic role of neutrophils in ischemic brain damage will likely remain uncertain. Moreover, the attempts of clinicians to modulate neutrophil trafficking to reduce brain damage in patients will continue to be compromised by the lack of clear insight and will remain more dependent upon intuition and conjecture than the solid empirical framework typically required for clinical success. ACKNOWLEDGMENTS The authors acknowledge the helpful comments of Drs. James Dasch and Susan Steitz-Abadi on an earlier draft of this review. Also, we appreciate to excellent assistance of Tom Jacobs, involving literature searches and manuscript preparation.

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