Brain inflammation is a frequent consequence of sepsis and septic shock. We imaged leukocyte recruitment in brain postcapillary venules induced by i.p. administration of LPS as a simple model of systemic inflammation. The i.p. injection of LPS (0.5 mg/kg) induced significant leukocyte rolling and adhesion in brain postcapillary venules of wild-type (WT) mice and more than 90% were neutrophils. However, no emigrated neutrophils were detected in brain parenchyma. High levels of TNF-α and IL-1β were detected in the plasma after LPS injection but a different profile (IL-1β but not TNF-α) was detected in the brain. LPS caused no recruitment in TLR4 knockout mice. In chimeric mice with TLR4-expressing resident cells but TLR4-deficient bone marrow-derived circulating cells, neutrophil rolling and adhesion was similar to WT mice. This observation is consistent with a requirement for resident cells in the LPS-induced neutrophil recruitment into brain microvessels. Transgenic mice engineered to express TLR4 exclusively on endothelial cells had a similar level of leukocyte recruitment in brain as WT mice in response to LPS. High dose LPS (10 mg/kg) led to neutrophil infiltration in the brain parenchyma in WT mice. High KC and MIP-2 production was observed from brain parenchyma microglial cells, and CXCR2 knockout mice failed to recruit neutrophils. However, neither neutrophil infiltration nor KC or MIP-2 was observed in endothelial TLR4 transgenic mice in response to this LPS dose. Our results demonstrate that direct endothelial activation is sufficient to mediate leukocyte rolling and adhesion in cerebral microvessels but not sufficient for emigration to brain parenchyma.
Sepsis and septic shock are leading causes of mortality in intensive care units worldwide. There is a growing body of evidence that systemic infection has a negative impact on CNS functions: Encephalopathy is a very common complication of patients with sepsis of different etiology, often leading to delirium and long-term cognitive impairment. Moreover, brain injury (1, 2) and neurodegenerative diseases including Alzheimer’s disease (3) and multiple sclerosis (4) could be significantly exacerbated by systemic infection. In animal experiments, systemic infection caused many physiological responses including changes in neuroendocrine and motor activity (5, 6, 7). Furthermore, systemic inflammation could also exacerbate brain inflammation (8) and increase neuronal death (9). Clearly, systemic inflammation can have a devastating effect on CNS function.
Leukocyte recruitment into the CNS is a key feature in many CNS diseases including stroke, multiple sclerosis, brain infection, and trauma (10, 11). Blocking leukocyte recruitment significantly benefited clinical courses of many CNS inflammatory conditions. Blood-brain barrier (BBB)2 limits immune cell trafficking into the CNS (12). Leukocyte recruitment occurs only following endothelial activation and is associated with disruption of the BBB. Whether endothelial activation and subsequent leukocyte recruitment is a direct effect of bacterial products such as LPS or an indirect effect of inflammatory cytokines released by microglia or macrophages remains unclear.
The systemic inflammatory response starts with recognition of bacterial products and the activation of different innate immune components including macrophages, neutrophils and endothelial cells. TLRs are a class of single membrane-spanning noncatalytic receptors that recognize structurally conserved molecules derived from microbes. To date, 10 human and 13 mouse members of the TLR family have been discovered (13). Recognition of bacterial products by TLRs causes direct cell activation leading to the expression of many immune factors such as proinflammatory cytokines and chemokines. However, whether it is direct activation of for example endothelial cells or microglia that leads to increased leukocyte recruitment remains unknown.
The objective of the present study was to investigate how systemic inflammation causes endothelial activation and neutrophil recruitment into the CNS. Systemic LPS induced significant neutrophil rolling and adhesion in CNS microvasculature but no parenchymal infiltration. Recruitment was dependent on TLR4 bearing residential cells but not on neutrophils or macrophages. Microglial activation was not required for neutrophil recruitment. In transgenic mice with TLR4 expression exclusively on endothelial cells (EndoTLR4), direct endothelial activation was sufficient for neutrophil recruitment. A high dose of LPS (10 mg/kg) caused neutrophil transmigration into CNS parenchyma in wild-type (WT) mice but not in EndoTLR4 transgenic mice or in microglia-inhibited mice. Transmigration into CNS required guidance from CXCL chemoattractants such as MIP-2 and KC that was not produced in EndoTLR4 mice. Overall, our findings demonstrate an essential role of direct endothelial activation inducing neutrophil recruitment into the CNS vasculature in systemic inflammation but emigration was dependent on other parenchymal cells.
Materials and Methods
Animal and reagents
C57BL6/J mice (referred to as WT) were purchased from Charles River Breeding Laboratories, TLR4 knockout (TLR4−/−) mice and CD14 knockout (CD14−/−) mice were purchased from Jackson ImmunoResearch Laboratories. EndoTLR4 transgenic mice were made in our laboratory as previously described (14). All the mice used in this study weighed 20–25 gram and were older than 8 wk. All the animal protocols were approved by University of Calgary animal care committee. Ultrapure LPS (Escherichia coli O111:B4) was obtained from LPS List Biological Laboratories, which never caused biologic response in TLR4 knockout mice.
Quantification of P-selectin expression level
P-selectin expression was measured as an index of endothelial activation using a modified dual radio-labeled Ab method as previously described (15). The Abs RB40.34 (specific to P-selectin) and A110-1 (rat IgG1 isotype) were labeled with I125 and I131, respectively. To measure the P-selectin expression, mice were injected with a mixture of 10 μg of 125I-labeled RB40.34 and a variable amount of 131I-labeled A110-1. The Abs were allowed to circulate in the body for 5 min, then the mice were perfused by saline. Tissues were harvested and weighed, and 125I and 131I activity was measured. P-selectin expression per gram of tissue was calculated by subtracting 131I-labeled A110-1 activity from 125I-labeled RB40.34. It has been previously demonstrated that this approach provides reliable and exquisitely sensitive results. Basal levels of P-selectin can be detected in untreated WT mice when compared with true zero values in P-selectin knockout mice.
Intravital microscopy of the brain microcirculation was performed as previously described (16, 17). Briefly, mice were anesthetized and craniotomy was performed using a high-speed drill (Fine Science Tools), and the dura mater was removed to expose the underlying pial vasculature. To observe leukocyte endothelial interactions, circulating leukocytes were fluorescently labeled by i.v. injection of rhodamine 6G. Leukocyte endothelial interactions in the brain microcirculation were observed using an intravital microscope (Axioskop; Carl Zeiss Canada) outfitted with a fluorescent source. All the experiments were recorded for playback analysis. Rolling cells were defined as cells that rolled at a speed slower than that of blood flow. Cells were considered as adhering when they stayed stationary for at least 30 s.
Measurement of blood flow velocity
RBC velocity within cerebral postcapillary venules was determined via analysis of the velocity of 1-μm diameter fluorescent microspheres (yellow/green, PolyFluor; Polysciences) injected i.v. Beads were visualized via epifluorescence (excitation: 450–490 nm; emission: 515 nm) (18). Video sequences of microspheres in the bloodstream were recorded via Volocity and analyzed with ImageJ. Perfusion velocity was determined by quantification of the velocity of 20 individual beads per vessel.
Generation of TLR4 chimeric mice
Bone marrow chimeras were generated by transferring bone marrow cells between TLR4 knockout mice and WT mice. Briefly, recipient mice were first irradiated with two doses of 500 rad (Gammacell 40, 137Cs gamma-irradiation source) on a 3-h interval, then 8 × 106 bone marrow cells from donor mice were transferred via the tail vein of recipient mice. For the next 8 wk, the mice were kept in a clean environment and fed with water with 0.2% neomycin to allow full immune reconstitution. Our previous study has confirmed that this protocol created a 99% reconstitution rate from donor bone marrow cells using Thy1.1 and Thy1.2 congenic mice (20). TLR4−/− bone marrow placed into TLR4−/− mice as a negative control never elicited a response different from TLR4−/− mice.
Animals injected with LPS or saline were deeply anesthetized with a mixture of ketamine and xylazine and perfused through the heart with ice-cold 10% formalin. Brains were removed and fixed in 10% formalin for 1 wk. Thick coronal sections (∼2 mm) were taken at about −1.0-−3.0 mm from Bregma. Formalin-fixed tissues were embedded in paraffin and then cut 5-μm thick using a cryostat (CM3050; Leica) and stained with dichloroacetate esterase to identify extravasated neutrophils.
Statistical analysis was performed with Sigma-Stat software. Values are expressed as the mean ± SEM. Differences between the two groups were analyzed by Student’s t test and were considered significant at p < 0.05.
The i.p. treatment of LPS caused systemic inflammation and activation in multiple organs
We i.p. treated WT mice with LPS (0.5 mg/kg body weight). Significant plasma levels of proinflammatory cytokines including TNF-α and IL-1β were detected after LPS injection. Concentrations of TNF-α reached over 2000 pg/ml in 1 h after injection and decreased thereafter (Fig. 1⇓A). Unlike TNF-α, IL-1β secretion peaked at 4 h (Fig. 1⇓B). At 24 h after LPS treatment, both TNF-α and IL-1β levels returned to basal levels. In contrast to the periphery, TNF-α levels in the CNS (Fig. 1⇓C) did not show a significant increase after LPS injection, whereas high levels of IL-1β were detected in the CNS 4 h after LPS systemic administration (Fig. 1⇓D). As a positive control, intracerebroventricular injection of LPS caused a similar increase in IL-1β to i.p. administration of LPS. Intracerebroventricular injection of LPS induced a very large increase in TNF-α, suggesting the brain has the capacity to produce this cytokine but does not do so following systemic LPS.
P-selectin expression was used as an index for endothelial activation. All organs examined including brain and spinal cord revealed significant activation of endothelium. The P-selectin level in peripheral organs including lung (Fig. 2⇓A) and heart (Fig. 2⇓B) significantly increased 4 h after systemic LPS administration. P-selectin expression in the brain (Fig. 2⇓C) and spinal cord (Fig. 2⇓D) was also elevated significantly but was an order of magnitude less than in peripheral organs. This level of expression is entirely consistent with an order of magnitude less leukocyte trafficking in brain vs other organs (see below).
Systemic LPS treatment caused leukocyte recruitment to the CNS microcirculation
Leukocyte recruitment in the brain was directly measured using intravital microscopy. Saline treatment caused no leukocyte endothelial cell interactions in the cerebromicrovessels (Fig. 3⇓A), whereas systemic treatment with LPS caused significant leukocyte endothelial interactions (Fig. 3⇓B) at 4 h. Leukocyte rolling increased further at 24 h (Fig. 3⇓C), whereas leukocyte adhesion reached peak levels at 4 h and remained elevated at 24 h (Fig. 3⇓D). LPS treatment decreased blood flow velocity by ∼30% (Fig. 3⇓E), which might help leukocytes better interact with cerebral endothelium. Interestingly, although circulating leukocyte counts were decreased by more than 80% after LPS treatment (Fig. 3⇓F), leukocyte recruitment in CNS postcapillary vessels was always evident, raising the possibility that the increased recruitment contributed to reduced peripheral circulating counts.
To confirm the type of cells being recruited into the brain vessels, we depleted neutrophils by injecting RB6-8C5 (anti-Gr-1) Ab at 24 h before LPS treatment. We previously demonstrated that this procedure could deplete 98% of neutrophils in the circulation without affecting other populations of cells (18). RB6-8C5 treatment resulted in a significantly diminished number of rolling (Fig. 3⇑G) and adherent (Fig. 3⇑H) leukocytes induced by systemic LPS treatment, indicating that rolling and adhering cells induced by LPS treatment were mainly neutrophils. Neutrophil depletion did not significantly reduce plasma TNF-α or IL-1β (data not shown), suggesting these cells were not the main source of the inflammatory cytokines.
Neutrophil recruitment is dependent on resident cells bearing TLR4
TLR4 and CD14 knockout mice were treated with LPS i.p. TLR4 knockout mice had no rolling or adhesion response to LPS treatment, whereas CD14 knockout mice showed neutrophil rolling (Fig. 4⇓A) and adhesion (Fig. 4⇓B) similar to WT mice. This response is consistent with previously reported TLR4-dependent, CD14-independent cellular responses in some cells (21). Proinflammatory cytokine TNF-α (Fig. 4⇓C) and IL-1β (Fig. 4⇓C) levels in the plasma were also measured in these mice. Interestingly, CD14 knockout mice had significantly reduced levels of both cytokines. Clearly, the cerebral vasculature responds to LPS in a TLR4-dependent but CD14-independent fashion, whereas systemic cytokine production is dependent on TLR4 and CD14. This suggests multiple cell types are involved in the response.
To determine which type of cells are important for inducing neutrophil recruitment to the brain vessels, chimeric mice were made by transplanting bone marrow cells between TLR4 knockout and WT mice. Irradiated TLR4 knockout mice reconstituted with WT bone marrow cells were termed C57→TLR4 mice and would have WT bone marrow-derived cells including macrophages and neutrophils but TLR4-negative residential cells including microglia, mast cells, astrocytes, neurons, and endothelial cells. Irradiated C57 BL6/J mice receiving TLR4-deficient bone marrow were termed TLR4→C57 mice. TLR4→C57 chimeric mice showed no decrease in either rolling or adhesion compared with C57→C57 chimeric mice (Fig. 5⇓A). By contrast, C57→TLR4 mice displayed significantly reduced adherent leukocytes compared with control chimeric mice and TLR4→C57 mice (Fig. 5⇓B). Clearly, activation of TLR4-positive residential cells such as microglia, mast cells, neurons, and endothelial cells were important for neutrophil adhesion. Interestingly, no chimeric approach was sufficient to reduce leukocyte rolling, suggesting multiple cell types induce this response.
It has been reported by others that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates via P-selectin (22). Despite our data that bone marrow-derived cells including platelets were not involved, we determined the role of platelets in neutrophil rolling and adhesion. We pretreated mice i.p. with rabbit anti-mouse thrombocyte serum (Accurate Chemical and Scientific) for 4 h before intravital microscopy. Platelet-depleted mice displayed similar levels of rolling and adhesion to LPS-treated WT mice (data not shown).
Leukocyte recruitment is not dependent on activation of microglia
Microglia are innate immune cells located in the CNS of myeloid origin expressing a wide array of TLRs. It is becoming increasingly clear that microglial activation plays an essential role in the host defense in the CNS. Recently, we have published a study showing that LPS administration directly into the brain caused very significant increases in TNF-α production by microglia. Moreover, in the model of intracerebroventricular LPS injection from our group, neutrophil recruitment was entirely dependent on microglia because addition of the potent inhibitor of microglial activation (minocycline) blocked neutrophil recruitment (16).
We hypothesized that activation of residential cells in the CNS including microglia caused leukocyte recruitment in response to systemic LPS. We separated mononuclear cells from the brain and performed intracellular staining to test for microglial activation. A very subtle increase in microglial activation was noted after systemic LPS treatment. Minocycline did not block neutrophil rolling (Fig. 6⇓A) or adhesion (Fig. 6⇓B) caused by systemic inflammation but previously blocked intracerebroventricular LPS injection-induced neutrophil recruitment (16). Clearly, a striking difference in mechanism exists between local and systemic LPS-induced neutrophil recruitment into brain vasculature.
Direct endothelium activation was sufficient for neutrophil recruitment into CNS vessels
The endothelium can be directly activated by LPS via TLR4. We made a number of transgenic mouse lines that expressed TLR4 exclusively on endothelium and chose one with similar amounts of receptor and response as WT endothelium. The characterization of this strain and the fact that there is TLR4 only on endothelium was published elsewhere (14). We treated these EndoTLR4 transgenic mice with LPS i.p. and noted significant endothelial activation from control values, but less than values induced in WT mice (Fig. 7⇓A). We next tested whether this degree of endothelial activation was sufficient to induce neutrophil recruitment. The number of rolling and adhering neutrophils in the brain microvasculature induced by systemic LPS administration was nearly identical in WT and EndoTLR4 transgenic mice (Fig. 7⇓, B and C). Thus the 50% decrease in P-selectin expression did not translate into less neutrophil recruitment in EndoTLR4 transgenic mice. Also, exclusive activation of brain endothelial cells by LPS in vivo was sufficient to induce neutrophil recruitment into the brain postcapillary venules. This occurred in the complete absence of proinflammatory cytokines in the plasma (Fig. 7⇓, D and E), suggesting that leukocyte recruitment into the CNS vessels was not dependent on increased levels of TNF-α and IL-1β during systemic inflammation. It was clear that LPS was able to directly activate the endothelium to induce neutrophil recruitment.
To further examine neutrophil infiltration in the CNS, we performed esterase staining. Systemic administration of a low dose (0.5 mg/kg body weight) of LPS used in this study only caused neutrophil endothelial interactions in the cerebral microvasculature. No neutrophil infiltration was noted in the CNS parenchyma. Therefore, we tried much higher doses of LPS (2 or 10 mg/kg body weight) which did lead to neutrophil recruited into the CNS parenchyma in WT mice. However, the increased dose of LPS did not induce neutrophil recruitment into the CNS parenchyma of EndoTLR4 transgenic mice (Fig. 8⇓A), indicating direct endothelial activation is not sufficient to cause neutrophil transmigration into CNS. We hypothesized that higher doses of LPS must have caused productions of neutrophil chemoattractants to induce neutrophil recruitment. MIP-2 and KC are chemoattractants for neutrophil recruitment, MIP-2 has been reported previously to mediate neutrophil recruitment into lung in response to LPS (23). Level of MIP-2 (Fig. 8⇓B) and KC (data not shown) were significantly increased in the brain homogenate in WT mice; however, very little MIP-2 activity was detected in the EndoTLR4 transgenic mice. We injected the high dose of LPS (10 mg/kg body weight) into CXCR2 knockout mice, in which responses to MIP-2 and KC are completely blocked. Interestingly, although these CXCR2 knockout mice displayed a similar level of neutrophil adhesion (Fig. 8⇓C) as WT mice in response to high dose LPS, there was no neutrophil infiltration observed in the brain parenchyma (Fig. 8⇓D), suggesting CXCL signaling is essential for neutrophil transmigration. Furthermore, minocycline, an inhibitor for microglial activation, efficiently inhibited MIP-2 levels in the brain (Fig. 8⇓E), indicating microglia are the main source for MIP-2. Overall, these data suggest that the entry of immune cells into CNS is controlled by chemokine signaling from parenchymal cells.
Systemic infection often has a devastating impact on the CNS. The recruitment of leukocytes in the CNS microvasculature is a very common phenomenon in many CNS diseases. We used intravital microscopy to investigate the mechanism of leukocyte recruitment in the cerebral microvasculature induced by systemic administration of endotoxin. LPS injection induced significant expression of proinflammatory cytokines in the circulation with more subtle increases in some (IL-1β) and not other (TNF-α) cytokines in the brain. In fact, the most likely source of these cytokines would be microglia, which are resident immune cells of myeloid origin in the CNS. Microglia express a wide array of TLRs (24), allowing their activation in response to different innate stimuli.
Our previous study has shown that microglial activation plays an essential role in detecting bacterial product in the brain parenchyma thereby inducing endothelial activation and subsequent neutrophil recruitment into CNS (16). However, very limited microglial activation and TNF-α could be detected after systemic treatment with LPS. Although systemic circulating TNF-α was very high, TNF-α was unable to leak into the brain. Interestingly, it was not simply a case of no access of LPS to the CNS parenchyma because IL-1β was increased in the brain to a greater degree than in the systemic circulation, and to a similar level as when LPS was injected intraventricularly. Minocycline, a specific inhibitor for microglial activation could not inhibit the neutrophil rolling and adhesion induced by systemic LPS. No neutrophil infiltration was observed in the CNS parenchyma in response to a low dose of LPS. These data are strikingly different from our previous work in which we injected LPS directly into the cerebroventricle and noted a microglial-dependent increase in TNF-α and IL-1β, which led to profound neutrophil rolling, adhesion, and transmigration into CNS parenchyma.
It remains unknown whether neutrophil recruitment into CNS vasculature is caused by direct endothelial activation or as a secondary effect from proinflammatory cytokines. Patients with multifocal necrotizing leukoencephalopathy in septic shock had much higher levels of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the plasma compared with healthy subjects (25). Although the endotoxemic mice also had elevated plasma cytokine levels, two pieces of evidence suggest that LPS can activate the CNS endothelium independent of signaling from inflammatory cytokines. First, robust neutrophil recruitment was observed at the 24-h time point, whereas very little proinflammatory cytokines remained in the plasma and CNS parenchyma at that time. Second, although cytokine levels were significantly reduced in CD14−/− mice, the neutrophil recruitment was unimpaired. These two observations suggest that endothelial cells serve as a sentinel population of pathogen detectors and mediate neutrophil endothelial interactions in the brain independent of inflammatory cytokines in systemic inflammation.
To date, there is no efficient way to selectively inhibit endothelial activation. We engineered a transgenic mouse that had TLR4 expression exclusively on endothelial cells under the Tie-2 promoter to examine whether endothelial cells alone could contribute to neutrophil recruitment. When these EndoTLR4 mice were treated with systemic LPS, almost no increases in inflammatory cytokines were observed in the circulation, whereas robust neutrophil recruitment was observed in cerebral postcapillary vessels. Clearly, without inflammatory cytokines, direct activation of CNS endothelial cells in vivo is entirely sufficient to cause significant neutrophil recruitment into CNS microvessels. These data are also quite consistent with the view that the plasma cytokine levels were not contributing to the leukocyte recruitment. It is worth noting that systemic LPS and elevated cytokines cause profound neutropenia, which makes recruitment into muscle and other peripheral organs very difficult. It is therefore intriguing that there were ample rolling and adhering cells in the cerebral vasculature, suggestive of some as yet unknown selective brain specific recruitment mechanism. Alternatively, there may be a delay in emigration of adherent neutrophils in the brain vasculature, which would allow for neutrophil accumulation in brain vasculature.
Systemic administration of low dose LPS caused rolling and adhesion in the CNS vessels via direct endothelial activation. However, similar administration did not lead to neutrophil transmigration into CNS parenchyma. It has been shown that activation of human endothelial cells with LPS in vitro is sufficient to cause robust rolling, adhesion and ample transmigration of neutrophils across endothelium (26). Moreover, we previously reported that LPS administration into cremaster muscle of EndoTLR4 transgenic mice induced significant neutrophil emigration (14). In other words, endothelial cells can tell neutrophils to emigrate independent of other cells. Interestingly, whatever the mechanism involved in this process, it is not evident in cerebral endothelium, and a second signal from brain parenchyma is required. Residing at the interface between the circulation and the brain parenchyma, CNS endothelial cells are thought to function as a formidable barrier to not only soluble molecules but also leukocyte trafficking. Clearly, the endothelial activation is sufficient to bring neutrophils to the BBB but not sufficient to allow neutrophil transmigration into CNS parenchyma. However, very high doses of LPS led to endothelial activation and subsequent neutrophil influx into CNS parenchyma. Others have reported that this is accompanied by breakage of the BBB (27). However, no neutrophil infiltration was noted in the EndoTLR4 transgenic mice, suggesting that other cells via cytokines or chemokines are required for neutrophil transmigration. MIP-2 and KC are dominant chemokines for attracting neutrophils in inflammatory diseases. Significant levels of MIP-2 and KC were detected in WT mice but not in EndoTLR4 transgenic mice. High doses of LPS did not recruit neutrophils into CNS parenchyma in CXCR2 knockout mice, consistent with the view that MIP-2 released from activated parenchymal cells other than the endothelium was required for neutrophil transmigration into the brain parenchyma. Preliminary work suggests this release to be from the microglia because minocycline, a potent inhibitor of microglia, blocked MIP-2 production.
Overall, in this study, we show that cerebral endothelial cells played a prominent role as sentinel cells in detecting and responding to endotoxemia. Peripheral inflammation led to neutrophil patrolling on the BBB by direct activation of cerebral endothelial cells. However, direct activation of endothelial cells was not sufficient to induce neutrophil migration into the brain parenchyma. Higher doses of endotoxin broke the BBB and induced chemokine production in microglia to perhaps guide neutrophil transmigration.
We thank Lori Zbytnuik, Carol Gwozd, and Dean Brown for excellent technical assistance.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Address correspondence and reprint requests to Dr. Paul Kubes, Immunology Research Group, Department Physiology and Biophysics, University of Calgary, 3330 Hospital Drive, NW Calgary, Alberta, T2N 4N1 Canada. E-mail address:
↵2 Abbreviations used in this paper: BBB, blood-brain barrier; WT, wild type.
- Received April 29, 2009.
- Accepted August 9, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.