INTRODUCTION
Reductionist investigations have greatly advanced our understanding of the human biology of severe inflammation and infection. It is now appreciated that innate immune responses to pathogens occur largely via immunocyte recognition of molecular motifs and the subsequent activation of numerous kinase pathways. It is also evident that temporally overlapping pro- and anti-inflammatory signals are generated during such responses. Unfortunately, variability of the clinical phenotype frequently precludes real-time interpretation of such influences and limits opportunities for effective intervention.
THE ENDOTOXIN PARADIGM
We have learned a great deal about the initial human response to infectious challenge from the elective administration of endotoxin, a lipopolysaccharide (LPS) that is a major component of the gram-negative bacteria outer membrane. Several preparations of this reagent have been commonly used (1, 2), including, more recently, CCRE, lot 2, which, when administered over a dose range of 1 to 4 ng/kg, induces a characteristic inflammatory phenotype in virtually all subjects. Although there is some dose dependency within the elicited phenotype and biochemical features of the model, it remains to be determined to what extent genetic, gender, and other clinical variables contribute to this dose-related effect.
SYSTEMIC AND ORGAN SYSTEM RESPONSES
Systemic features
Within 1 h after the intravenous administration of endotoxin (LPS), subjects may variably experience symptoms ranging from chills, headache, back pain, myalgias, nausea, and photophobia. The expression of discomfort from these symptoms varies between subjects, although virtually all subjects report attenuation of symptoms within 4 to 6 h. The most reproducible features include increases in core temperature (1°C-4°C) and heart rate. These manifestations of systemic inflammation generally abate within 6 to 8 h.
Organ system changes
Much of what we have learned about the human biology of endotoxemia has arisen from observations in this model. In particular, confirmation of preclinical descriptions of cytokine activation was an early focus of these studies (3-8). The appearance of cytokines was found to follow a characteristic pattern, beginning with the TNF-α activity, which is found to be increased in the circulation within 45 to 60 min after LPS injection. The monophasic peak of TNF activity occurs within 2 h and is rapidly followed by a rise in soluble TNF receptor activity that may explain, in part, why no further TNF bioactivity is detectable thereafter (6). Ex vivo testing of whole blood samples within the 2- to 4-h time frame after in vivo LPS administration documents a diminished TNF responsiveness to endotoxin (9-11). Soluble factors appear responsible, in part, for this influence as plasma obtained 2 h after LPS challenge also suppresses TNF production by naive blood cells when challenged ex vivo with endotoxin. These observations replicate the ex vivo acute LPS hyporesponsiveness frequently observed in patients (12). This influence upon ex vivo cells is reversible within 6 to 24 h in normal subjects and during the recovery phase of patients. Interestingly, the subsequent readministration of LPS within 1 week after the initial challenge elicits neither symptoms nor a detectable in vivo TNF response (unpublished observations).
The peak of TNF activity is rapidly followed by appearance of other “pro-inflammatory” cytokines such IL-6 (13) and IL-8 (14, 15) (Fig. 1). The chemokines IFN-inducible protein 10, MCP-1, MIP-1α and β (16, 17), as well as the G-CSF (18) and IL-18 (19) are also detectable subsequent to the TNF activity peak. Soluble IL-1 species, with the exception of IL-1ra, are minimally detectable, although cell associated IL-1β has been noted (20).
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FIG. 1: Transient blood endotoxin levels are followed by the appearance of circulating pro- and anti-inflammatory cytokine mediators after low dose intravenous endotoxin administration (14, 25, 31).
The model of human endotoxemia also elicits a panoply of systemic counter-regulatory mechanisms ranging from neuroendocrine responses (see below) to cellular and soluble antagonists of proinflammatory activity. Soluble inhibitors of TNF and IL-1 activity are readily produced, including TNF type I and type II receptors (6, 21, 22) as well as the IL-1receptor antagonist (23, 24). Soluble IL-1 type II receptor levels do not rise after LPS administration (25).
EFFECTS ON IMMUNE CELLS
The human endotoxemia model elicits dynamic and reproducible changes in the circulating leukocyte population as well as the function of such cells. The circulating leukocyte count begins to decline within 15 to 30 min after LPS challenge (26) at a time before the onset of clinical symptoms or mediator appearance. This is noteworthy for a marked monocytopenia that reverts toward normal over the ensuing 6 to 8 h. A progressive decline in total lymphocyte count also begins shortly after LPS challenge and continues over the ensuing 9 to 12 h. The differential of the leukocyte count is influenced largely by the circulating polymorphonuclear (PMN) leukocyte population that, after an early decline, rises to levels above basal within 4 to 6 h and returns to normal within 24 h (Fig. 2).
FIG. 2: Changes in circulating leukocyte counts following low-dose intravenous endotoxin administration (26).
In addition to the hyporesponsiveness of immune cells to acute rechallenge with endotoxin, these cells also undergo changes in cell surface expression for cognate ligands of inflammation. The most dramatic of these changes involves an acute down-regulation of the type I and type II TNF receptors that occurs within 30 min of LPS administration and recovers fully over the ensuing 24 h (Fig. 3) (27). Interestingly enough, this dramatic alteration in TNF receptor expression is not accompanied by similar changes in receptors for endotoxin (toll-like receptor 4 or 2, TLR) or CD95 (APO-1, apoptosis receptor) (28). We have observed similar dynamics among at risk and severely infected patients (29).
FIG. 3: Changes in circulating monocyte expression of cell surface antigens following low-dose intravenous endotoxin administration (
27).
The endothelial cell can be included among the immune cell populations exhibiting activation after in vivo LPS challenge. As evidenced by surrogate markers, such as soluble E-selectin, robust endothelial cell responses can be observed within 2 h of LPS challenge and persist for over 6 h afterward. There is also a modest increase in whole-blood TF activity (30).
Hemostatic dysfunction
Among the many interesting correlates to human infectious pathology arising from the human model of endotoxemia is the documentation that low-dose LPS induces activation of the fibrinolytic and coagulation systems (31, 32). Interestingly enough, fibrinolytic activation, as assessed by release of tissue-type plasminogen activator (tPA) followed by a rise in plasminogen activator inhibitor type I (PAI-I) and plasmin-a2-antiplasmin complexes (PAP). The fibrinolytic system activity has largely abated within 3 to 4 h, at which time the activation of the coagulation system is continuing. Coagulation activation is observed to increase within 1 h and continues for up to 4 to 5 h as assessed by increases in prothrombin fragments (F1 + 2) and thrombin-anti-thrombin III complexes (TAT). Activation of the extrinsic pathway via tissue factor appears to be largely responsible for the net procoagulant state induced by endotoxemia.
Effects on organ system function
Cardiovascular function-
The hemodynamic effects of low-dose endotoxin infusion in healthy humans have been well documented (1-3, 20, 33). Several studies have observed a hyperdynamic cardiovascular response in association with increased cardiac index and as well as modest decreases in peripheral vascular resistance. These changes are of limited duration (<6 h). All parameters of cardiovascular function return to normal within 12 to 24 h. There have been no reports of severe cardiovascular complications resulting from endotoxin administration to young, healthy subjects.
Despite attendant tachycardia, endotoxin administration also elicits a diminution of low frequency and very low frequency variation of heart rate (34) during the first several hours after infusion. This interesting observation is similar to that noted in critically ill patients (35). Although the mechanism for this loss of variability (decomplexification) is unknown, it is likely that altered central autonomic activity and/or imbalance of sympathetic and parasympathetic efferent signaling are contributory.
Pulmonary function
Although there is no documentation that low-dose endotoxin alters respiratory gas exchange (4), some evidence points to a modest increase in lung permeability to small molecules but not to larger proteins. Bronchoalveolar lavage (BAL) after low-dose systemic endotoxin challenge does not reveal increased influx of PMNs although one study has observed a transient priming of BAL obtained macrophages when these cells challenged with LPS ex vivo (5).
Several studies have used low-dose endotoxin administration directly into the pulmonary tree by aerosolized inhalation or direct segmental installation (36). Studies of inhaled LPS reveal a dose-response relationship with respect to pulmonary and systemic inflammatory responses (37).
Other organ functions
Endotoxin administration has been reported to increase intestinal permeability in healthy subjects. Excretion of orally administered lactulose, a marker of gut barrier function, is increased 2-fold during the 12-h period after endotoxin challenge (38). The magnitude of this purported barrier disruption also correlated directly with catecholamine levels and inversely with the neutropenia response.
Additional, albeit indirect, evidence of altered gut barrier function derives from a study of normal subjects who had been fed exclusively by the parenteral route for the previous 10 days. In response to endotoxin, enhanced proinflammatory cytokine responses (39), interorgan substrate flux (22), and acute-phase reactant levels (39) are observed in subjects with previous bowel rest. This study suggests that antecedent gut-derived (endotoxin) priming as well as acutely altered barrier function may occur in this setting.
Neuroendocrine responses
A classical pattern of stress hormone secretion results from low-dose endotoxin injection (7, 8, 40, 41). This includes an early spike in ACTH followed by a later increase in cortisol over 3 to 4 h after endotoxin injection. An increase in catecholamine (epinephrine and norepinephrine) secretion is also evident within 1 to 2 h and abates over the ensuing 4 to 6 h. Corticotropin-releasing hormone is not reported to increase.
Hormones regulating substrate and energy metabolism are also altered by LPS infusion, including an early increase in growth hormone, a gradual decrease in insulin-like growth factor-1, and later increases in IGF-1-binding proteins 1 and 2 (42). Thyroid hormone levels are also altered, including reduced thyroxine (T4) and triiodothyronine (T3) levels, and increased reverse T3 levels (43, 44). The acuity of such changes in the absence of antecedent abnormalities of thyroid metabolism or of malnutrition suggests a potential role of endotoxin (or of endotoxin-inducible mediators) in the evolution of the euthyroid sick syndrome.
Metabolic responses
Given the above changes in endocrine hormone influence, it is not surprising that significant alterations of intermediary metabolism and of interorgan substrate flux also accompany low-dose endotoxin infusion. An increase in whole body energy expenditure is observed within 2 h and is coincidental with the initial evidence of proinflammatory mediator production and catecholamine secretion (24, 45). Splanchnic oxygen consumption also rises during this period (40). This change generally persists through the period of pyrexia and returns to normal within 8 to 24 h.
In the absence of significant glucose counter-regulatory hormone (insulin and glucagon) changes, blood levels of glucose and lactate are little altered although whole body isotope studies (22, 40, 46) suggest increased glucose turnover. Our studies of splanchnic glucose flux demonstrate increased hepatic lactate uptake and glucose production consistent with enhanced gluconeogenesis (40). Increased hepatic uptake of gluconeogenic amino acids (alanine and glutamine) is also observed. We have also documented increased whole body protein breakdown and peripheral tissue net amino acid release (and pattern of amino acid efflux) consistent with skeletal muscle proteolysis (22).
Lipid metabolism is also altered, as evidenced by an increase in circulating free-fatty acid levels over 2 to 6 h after LPS injection and a net efflux of FFAs from peripheral tissues. A cotemporaneous increase in hepatic uptake of free-fatty acids and release of triglycerides is also observed (40). Acute or latent influences on cholesterol and lipoprotein levels have not been thoroughly investigated nor has the relationship of antecedent lipoprotein pattern upon LPS responsiveness.
MODIFICATION BY CLINICAL CONDITIONS AND THE IMPLICATIONS OF COMPLEX DISEASE
There are numerous conditions of clinical relevance that influence the responsiveness of normal subjects to endotoxin administration. These interactions may influence the salient response features of the model and mechanistically driven therapeutic regimens amongst broader endotoxemic populations.
Humans exhibit diurnal susceptibility to endotoxin. This has been demonstrated after intravenous challenge at 9:00 AM and 7:00 PM using Salmonella abortis equi endotoxin (0.8 ng/kg) (47). Although the time of day did not influence the appearance of TNF-α or IL-6, subjects receiving infusion at 7:00 PM demonstrated a near doubling in core temperature and ACTH/cortisol levels compared with morning administration. This suggests that the phenomenon of diurnal response variation is, at least partially, independent of direct proinflammatory cytokine mediation.
Antecedent hypothalamic-pituitary adrenal axis activation influences phenotypic and biochemical features of human endotoxemia. This has been conclusively proven by studies evaluating responses within the context of existing stress hormone excess. Glucocorticoid excess, as produced by hydrocortisone injection (100-200 mg) (50) or 6-h infusion (3 μg/kg/min) (7) before LPS challenge abrogates much of the clinical symptoms (including temperature, heart rate, and increased energy expenditure), as well as proinflammatory cytokine and cytokine antagonist appearance associated with naive injection (40). Antecedent hypercortisolemia (>6 h) also increases IL-10 levels and diminishes activation of the coagulation and fibrinolytic pathways (49).
Interestingly enough, the attenuation of endotoxin responses to hypercortisolemia is relatively short lived. Indeed, the return of characteristic phenotypic and biochemical features of low-dose LPS challenge return quickly in the face of ongoing cortisol excess (>12 h), and heightened TNF-α levels are actually observed after extended periods of eucortisolemia (144 h) before endotoxin injection (7).
Altered rates of spontaneous and CD95 (APO-1) ligand-induced PMN apoptosis are also a characteristic feature of this model. Circulating PMN exhibit significantly diminished apoptosis after in vivo LPS exposure, and this cellular (dys) function persists for up to 72 h after endotoxin challenge (50). Six hours of cortisol excess before LPS challenge does not alter the acute reduction in PMN apoptosis (initial 1-12 h), but does promote earlier restoration of spontaneous and inducible PMN programmed cell death (<24 h; Fig. 4).
FIG. 4: Six hours of cortisol excess (HC) prior to LPS challenge does not alter the acute reduction in PMN apoptosis (initial 1 to 12 h) but does promote earlier restoration of both spontaneous (CH11−) and inducible (CH11+) PMN programmed cell death (<24 h) (50).
Catecholamine excess also alters several features of the human endotoxin model. When administered (30 ng/kg/min) for periods of 3 or 24 h before LPS challenge, the appearance of TNF-α was diminished. This effect was more pronounced with the shorter infusion period, which was also notable for enhanced IL-10 release (51). This catecholamine infusion regimen is also remarkable for a significant attenuation of net coagulation pathway activation (Fig. 5) (52).
FIG. 5: Epinephrine inhibits LPS-induced coagulation activation (assessed by thrombin antithrombin complex). LPS = subjects injected with LPS only (n = 6); EPI = subjects infused with epinephrine (30 ng/kg/min) from t = −3 h to 6 h (n = 5); EPI-24 = subjects infused with epinephrine (30 ng/kg/min) from t = −24 h to 6 h (n = 6). • = LPS, ○ = EPI-3, □ = EPI-3 (52).
Nutritional status influences
As noted earlier, studies have also documented that the route of feeding also influences human responses to endotoxin. Normal subjects who have received a 1-week period of total parenteral nutrition (nothing by mouth) exhibit a significantly greater inflammatory phenotype (heart rate and temperature) as well as systemic and splanchnic production of TNF-α and CRP than do subjects from an orally fed cohort (39, 45). This enhanced inflammatory response is associated with enhanced peripheral tissue (extremity) amino acid and lactate efflux, suggesting that intravenous feeding predisposes such tissues to endotoxin-inducible catabolic stressors. It remains to be determined if this tendency arises from the cumulative effects of immobility and loss of diurnal anabolic hormone influences and/or from the acute influence of excess proinflammatory mediator activity. Intriguingly, the intravenous feeding route also induced increased coagulation activation in response to endotoxin but no differences exist at baseline (Fig. 6) (53).
FIG. 6: Activation of coagulation. Mean (±SE) plasma concentration of thrombin-antithrombin III (TAT) complexes after intravenous injection of endotoxin (ng/kg) at t = 0 in healthy humans who had received total parenteral nutrition or normal enteral feeding one week before endotoxin administration (53).
The above studies were performed under conditions of glucose-based nonprotein calorie support. We have also tested the hypothesis that nonprotein energy background may also alter endotoxin responses. The acute administration of a high-density lipid emulsion (20%) increases inflammatory cytokine (IL-6 and IL-8) appearance and also enhances coagulation activation (54). These interesting observations suggest that the route of nutritional support as well as the mix of substrate provision may be consequential to the evolving features of acute endotoxemia. Such observations have clear implications for the management of patients at risk for infectious complications.
Molecular influences
The clinical phenotypes of severe infection are very likely modulated by genetic and environmental (life-style and disease comorbidity) factors. These nuances are being approached with sophisticated tools of molecular discovery and diagnosis. In concert with colleagues participating in a multi-institutional National Institutes of Health-funded “Host Response to Inflammation and Injury” (U54GM62119) grant, we have begun to investigate the whole blood immune cell response to endotoxin injection. The first generation of these studies has used a unique RNA isolation method that exhibits fidelity within individuals as well as significant interindividual correlations among gene expression patterns at postendotoxin time points.
We and others have also begun to use the human endotoxin model in an approach to the assessment of single nucleotide polymorphisms (SNPs) that have been ascribed to altered risk and outcome in seriously ill patients (55). The performance of these studies in healthy, younger subjects provides a clearer snapshot of the LPS-induced phenotype in the absence of expressed comorbidities or injury conditions.
The analysis of mutations within the TLR4 gene provides an example of the potential for confounding influences of comorbidities, and factors such as age, gender, and injury, upon the ultimate clinical phenotype. Because the original description of the 299 G-A (and the cosegregating 399 T-C) mutation of TLR4, there have been several reports suggesting that these SNPs alter risk and outcome for gram-negative infection (56). This causal relationship is by no means established, as neither in vitro studies of cells from subjects bearing these mutations nor other clinical studies have observed altered LPS responsiveness or altered risk/outcome. Studies of LPS administration to humans have yielded similarly disparate results, with the inhalation of ligand demonstrating attenuated systemic responses (37) in subjects bearing these SNPs, whereas the intravenous LPS challenge model suggests no influence of the 299/399 TLR4 mutations (55).
USE IN BIOLOGIC MODIFICATION
The human endotoxemia model provides a test platform to assess proof of principle for agents directed against activity of an LPS ligand as well as for interdiction against LPS-inducible signaling pathways and mediators. There have been several studies reported, including some in which the prophylactic interdiction against the endotoxin ligand have proven very successful in attenuating the phenotypic manifestations and cytokine release attending endotoxin challenge (57). Other strategies directed against the ligand have shown more variability with respect to clinical phenotype, but readily detectable influences upon inducible cellular and mediator responses (3, 58-61).
Therapeutic strategies designed to alter the endocrine and paracrine activity of endotoxin-induced cytokines have documented the complex dose-response pharmacology (33) and, sometimes unanticipated, consequences of these approaches (20). Such studies are instructive from a mechanistic and therapeutic perspective. There have also been efforts to alter the proinflammatory consequences of endotoxin challenge by administration of hydrocortisone (7, 62) and IL-10 (63), with both approaches demonstrating attenuation of inflammatory responses only when administered before endotoxin challenge. Other studies have sought to modulate endotoxin-induced inflammatory responses by antecedent administration of G-CSF, again documenting the complexities of dose-timing interactions (18, 64).
The appealing concept of targeting endotoxin-sensitive biochemical pathways has been investigated in several studies. This includes the less selective approaches to cyclooxygenase blockade (15, 65, 66), which proved of modest benefit albeit with some unanticipated enhancement of cytokine release, and the administration of pentoxyphilline, which demonstrated no significant influence (15, 67). Interestingly enough, recent studies of p38 MAP kinase blockade demonstrate rather dramatic anti-inflammatory effects of this selective approach (68). Doubtless, this more precise approach to signaling pathway interdiction will prove a useful application of the model as will efforts to alter cellular energetics.
The current concept that excessive coagulation activation underlies some of the pathophysiology of severely endotoxemic patients has led to several recent studies addressing the influence of agents affecting coagulation. Among these are studies of heparins (69), as well as those using tissue factor pathway inhibitor (70) and activated protein C (71, 72). By and large, these studies have not documented an influence of these agents upon the clinical phenotype or the release of inflammatory mediators or markers of cellular activation. This suggests several possibilities, including a limited anti-inflammatory effect of these agents or a limitation of a model that lacks previous stress or factor deficiencies.
These latter studies address some of the caveats attending the use of the human endotoxin challenge model for proof-of-principle purposes. Any expectation that the model fully replicates the clinical condition of severe, localized or systemic gram-negative infection is unwarranted. The relatively brief temporal sequencing of pro- and anti-inflammatory responses simulates neither the smoldering nor highly dramatic clinical responses induced by many infections. Furthermore, no interventions are required to fully resolve the phenotypic consequences of LPS challenge nor are the confounding influences of associated injury, disease comorbidity, or therapy at play.
Despite these shortcomings, this model has use in determining whether a given experimental approach modifies the nonspecific inflammatory phenotype (SIRS). Previous experiences in this model suggest that the absence of this effect portends minimal therapeutic influence of a given strategy or agent in lower risk clinical conditions. The model may also provide insights about untoward consequences that are not readily obtained from preclinical studies.
SUMMARY
Human models that administer endotoxins provide mechanistic insights into how competent cells and organ systems respond to these ligands. The model transiently alters many systemic physiologic and metabolic processes in a qualitatively similar manner to the early flow phase of injury and infection. Recent data also suggests that early transcriptional profiles are similar (but not identical) to those observed in injured patients. There is great interest in dissecting the relevant differences between the no-risk phenotype and molecular profile of transient human endotoxemia compared with more complicated and risk prone clinical conditions (73). This challenge is all the more daunting within the clinical context of influences exerted by genetic variation, sexual dimorphism, age, and chronic illness.
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49. van der Poll T, Barber AE, Coyle SM, Lowry SF: Hypercortisolemia increases plasma interleukin 10 concentrations during human endotoxemia.
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50. Lin E, Calvano SE, Coyle S, Rumalla V, Kumar A, Lowry SF: Physiologic hypercortisolemia in humans modulates neutrophil CD95-signal transduction.
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51. van der Poll T, Coyle SM, Barbosa K, Braxton CC, Lowry SF: Epinephrine inhibits tumor necrosis factor α and potentiates interleukin 10 production during human endotoxemia.
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52. van der Poll T, Levi M, Dentener M, Jansen PM, Coyle SM, Braxton CC, Buurman WA, Hack CE, ten Cate JW, Lowry SF: Epinephrine exerts anticoagulant effects during human endotoxemia.
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