We then tested the ability of the soluble 12-amino acid mimic, N2, to change these parameters of HS. This agent is the exact sequence of the binding site of the pathogenic natural IgM to ischemic tissue and prevents binding of IgM by occupying the IgM ligation sites in the fluid phase (18, 19), in effect, a decoy ligand. The agent, thus, is specific for reperfusion injury. Three hundred micrograms was selected as the dose based on the effective dose in mice (18, 19) multiplied by mass of rat/mass of mouse, as if a rat were a large mouse. Although smaller doses of N2 are known to be effective pretreatment for reperfusion injuries (18, 19) and burns (20), this timing of administration was chosen to mimic the clinical scenario of resuscitation from trauma. Administration of 300 μg N2 peptide with the i.v. return of shed blood at the end of the shock period improved survival at 72 h from 38% (3 of 8) to 88% (7 of 8, P < 0.05; Fig. 8). In addition, gut injury score at 72 h improved from 57.8% ± 5.5% in untreated animals to 19.5% ± 2.5% in N2-treated animals (P < 0.05; Fig. 1). The prominent IgM and C5b-9 deposition seen in shocked animals was no longer visible in N2-treated animals (Figs. 2 and 3). Likewise, pulmonary injury was attenuated by N2 treatment, with reduced neutrophil infiltration (21.4 ± 1.5 PMN per high-power field in untreated animals to 14.8 ± 1.3 in N2-treated animals [P < 0.05]; Fig. 5) and reduced capillary permeability (permeability index, 0.61 ± 0.14 in untreated animals to 0.18 ± 0.06 in treated animals [P < 0.05]; Fig. 6). Finally, we tested 300 μg of a control 12-amino acid peptide [Skeletal Muscle Myosin (SMM)] and saw only a slight effect on survival, gut injury score, or pulmonary permeability index. This peptide was, in fact, the sequence for the hinge region of skeletal muscle myosin, comparable in other respects to nonmuscle myosin. This lack of activity indicates a high degree of specificity to the action of N2 peptide (Figs. 1, 4-6, and 8).
We have chosen a simple model of resuscitated HS to test an agent specific for reperfusion injury and determine whether death and other effects caused by resuscitated shock is caused by global reperfusion injury. Another model of rodent HS using hemorrhage to a defined arterial pressure is more commonly in use because it produces measurable effects without death, the basic end point that we wished to examine. To that end, we titrated the shock time to produce 50% mortality at 72 h after resuscitation but no mortality during the shock period itself. It is important not to have loss of experimental animals during the injury phase of an experiment involving inflammation, as the animals lost could be those individuals with the most active proinflammatory systems. This is why we eliminated knockout mice as the method of study, as we have found significant differences in mortality from just the shock between relevant experimental strains (21); to study shock and resuscitation, the clinically relevant issue, all animals need to survive to resuscitation.
The findings on complement depletion by CoVF suggested that mortality in our model was influenced by the presence or absence of an intact complement system. However, a by-product of this depletion method is systemic generation of the complement anaphylatoxins, an event known clinically to impair neutrophil function (26, 27). Thus, results with this method of depletion can also be interpreted as reflective of neutrophil involvement. The sCR1 (6) is an agent, now in commercial development (TP10; Avant Therapeutics, Needham, Mass), that is an inhibitor of complement C3b, C4b, and C1q. It was used extensively in the early studies of reperfusion injury (6, 24, and many others) to prove the central role of complement in the pathogenesis of reperfusion injury. However, for our purposes, because sCR1 prevents generation of complement anaphylatoxins and, therefore, secondary pulmonary injury, use of this agent might not allow us to discriminate whether death from HS was caused by reperfusion injury or its secondary effects.
We chose instead to determine whether HS and resuscitation produced the typical appearance of a reperfusion injury in an organ, the intestine, thought to be particularly susceptible to such an injury. We found evidence of gut injury (Fig. 1) at 72 h in shocked animals, as quantitated by a scoring system that grades disruption of villi and lifting of the intestinal epithelium off of the mucosa. For this determination, 10 random sections in each animal were assessed. We also found that there was both complement (C5b-9) and IgM deposition on the villi of shocked animals (Figs. 2 and 3). Few neutrophils were evident. For this analysis, a 4-h time point was selected because later tissue necrosis may produce amplifying C3 and IgM deposition or loss of the target tissue altogether. In addition, less injured sections were used because it is impossible to assess deposition on villi that have completely disintegrated. Nevertheless, partial disruption of the tips of villi is visible clearly in the sections from shocked animals, a feature not present in sham-treated animals. This analysis strengthens the hypothesis from the CoVF experiment that complement-generated injury plays a role in HS. Furthermore, the presence of IgM, a hallmark of early reperfusion injury, indicated that the source of complement activation might be IgM deposition as a result of reperfusion injury (28). Thus, HS produces the typical features of reperfusion injury in the gut.
A second indication that a reperfusion injury might be present after HS is the development of a secondary pulmonary injury, a prominent feature of mesenteric reperfusion injury. Indeed, in this HS model, pulmonary neutrophil sequestration was produced (Figs. 4 and 5), as was increased pulmonary capillary permeability (Fig. 6). We observed a direct correlation between the severity of the gut injury and the severity of the pulmonary capillary leak (Fig. 7), suggestive of a causal link. Thus, HS might produce pulmonary injury as a result of reperfusion injury to the gut. However, this conclusion must be tempered by several other considerations: (a) We are reporting studies on only one zone of potential reperfusion injury. Other organs might be more severely affected and causing the pulmonary injury. (b) It is conceivable that the pulmonary injury itself is primary reperfusion injury and that the correlation represents the overall severity of HS on an individual basis.
The final indication that a reperfusion injury might be present is the inhibition of injury parameters caused by an agent specific to reperfusion injury. The development of this agent arose from a series of observations. First, gut and skeletal muscle reperfusion injuries were absent in antibody-deficient animals and were present if animals were reconstituted with IgM purified from normal mice (15, 16). Second, mice lacking in natural IgM antibody repertoire were as protected from reperfusion injury as were the completely antibody-deficient mice. Restoration of injury was possible by either reconstitution with normal IgM or transplantation of peritoneal B-1 lymphocytes (17, 29). Third, cloning of B-1 lymphocytes, the source of natural IgM, produced a single clone of IgM that was capable of restoring reperfusion injury in antibody-deficient mice (10, 11). This implied that there was a specific antigenic target that appeared on ischemic tissue. This target was identified by interacting the pathogenic IgM clone with a 12-mer peptide phage display library and with the ischemic tissue itself. This yielded both the protein with which the IgM interacted and the site within the protein that was the antigen, nonmuscle myosin heavy chain. A series of similar 12-amino acid peptides was synthesized and tested for the ability to inhibit the interaction. The peptide that corresponds to the exact sequence in the parent protein, N2, inhibited reperfusion injury, whereas random peptides and even the peptide representing the hinge region from muscle myosin heavy chain did not (18, 19). Thus, N2 peptide acts at the initiation of reperfusion injury and not in its inflammatory amplification. As such, it is a highly specific inhibitor. We chose to administer N2 peptide in this HS model in real clinical time, that is, with resuscitation. Given in this way, N2 peptide prevented or nearly prevented all of the features that accompany HS that we assessed. Death rates were reduced (Fig. 8). Intestinal mucosal injury was reduced (Fig. 1), as was C5b-9 deposition and IgM deposition, the anticipated mechanism of action (Figs. 2 and 3). Pulmonary neutrophil sequestration and capillary permeability increases were attenuated (Figs. 4-6). Thus, an agent that is highly specific for reperfusion injury prevents death and stigmata of HS, suggesting that reperfusion injury causes the adverse consequences of HS in this model. The hypothesis that relates reperfusion injury to HS is not novel (3) nor is the attempt to prove this linkage or treat HS with agents developed from the study of reperfusion injury (30, 31). The novelty of these findings is the simplicity of the agent (12-amino acid peptide), the high degree of specificity for the pathway involved in generating a reperfusion injury, and the ability to treat in real clinical time (at resuscitation).
There is a contrast in effects noted between the treatment of mesenteric reperfusion injury with complement inhibition (sCR1) (24) and the treatment of HS with N2 peptide. In the former, although sCR1 prevented increased pulmonary capillary permeability, it did not prevent increased pulmonary leukosequestration, as assessed by tissue content of myeloperoxidase. The interpretation was that pulmonary injury, as manifested by permeability changes, required both pulmonary endothelial activation and neutrophil activation. The sCR1 treatment, by preventing the generation of complement anaphylatoxins systemically, prevented the pulmonary endothelial activation. But the neutrophils continued to be activated by passage through the vasculature of injured gut, resulting in leukosequestration without injury. However, in this HS model, N2 peptide prevented both effects. This could indicate that the pulmonary injury in this HS model is not purely secondary to reperfusion injury in other susceptible organs and that the N2 peptide suggests that there is a primary pulmonary reperfusion injury as a result of HS.
Finally, it should be noted that the N2 peptide was developed as a result of murine protein and antibody studies. This HS study is in rats. The sequence of the hinge region of nonmuscle myosin heavy chain is identical throughout all species studied from yeast to humans. Thus, N2 is potentially effective in many species, as long as the basic mechanism of injury causing exposure of this antigenic region to circulating antibodies is maintained.
1. Cotran RS: Robbins Pathologic Basis of Disease
. St. Louis, MO: Saunders, 7-12, 1999.
2. Hierholzer C, Billiar TR: Molecular mechanisms in the early phase of hemorrhagic shock. Langenbecks Arch Surg
3. Peitzman AB, Billiar TR, Harbrecht BG, Kelly E, Udekwu AO, Simmons RL: Hemorrhagic shock. Curr Probl Surg
4. Coimbra R, Melbostad H, Hoyt DB: Effects of phosphodiesterase inhibition on the inflammatory response after shock: role of pentoxifylline. J Trauma
5. Paxian M, Keller SA, Huynh TT, Clemens MG: Perflubron emulsion improves hepatic microvascular integrity and mitochondrial redox state after hemorrhagic shock. Shock
6. Weisman HF, Bartow T, Leppo MK, Marsh HC Jr, Carson GR, Concino MF, Boyle MP, Roux KH, Weisfeldt ML, Fearon DT: Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science
7. Hecke F, Schmidt U, Kola A, Bautsch W, Klos A, Kohl J: Circulating complement proteins in multiple trauma patients: correlation with injury severity, development of sepsis, and outcome. Crit Care Med
8. Heideman M, Kaijser B, Gelin L: Complement activation and hematologic, homodynamic, and respiratory reactions early after soft-tissue injury. J Trauma
9. Heideman M, Norder-Hansson B, Bengston A, Mollnes T: Terminal complement complexes and anaphylatoxins in septic and ischemic patients. Arch Surg
10. Zhang M, Austen WG Jr, Chiu I, Alicot EM, Hung R, Ma M, Verna N, Xu M, Hechtman HB, Moore FD Jr, et al.: Identification of a specific self-reactive IgM antibody that initiates intestinal ischemia/reperfusion injury. Proc Natl Acad Sci U S A
11. Austen WG Jr, Zhang M, Chan R, Friend D, Hechtman HB, Carroll MC, Moore FD Jr: Murine hindlimb reperfusion injury can be initiated by a self-reactive monoclonal IgM. Surgery
12. Chan R, Ibrahim S, Takahashi K, Kwon E, McCormack M, Ezekowitz A, Carroll MC, Moore FD Jr, Austen WG Jr: The differing roles of classical and mannose binding lectin complement pathways on the events following skeletal muscle ischemia-reperfusion. J Immunol
13. Jordan JE, Montalto MC, Stahl GL: Inhibition of mannose-binding lectin reduces postischemic myocardial reperfusion injury. Circulation
14. Hart ML, Ceonzo KA, Shaffer LA, Takakashi K, Rother RP, Reenstra WR, Buras JA, Stahl GL: Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent with involving C1q. J Immunol
15. Weiser MR, Williams JP, Moore FD Jr, Kobzik L, Ma M, Hechtman HB, Carroll MC: Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement. J Exp Med
16. Williams JP, Pechet TTV, Weiser MR, Reid R, Kobzik L, Moore FD Jr, Carroll MC, Hechtman HB: Intestinal reperfusion injury is mediated by IgM and complement. J Appl Phys
17. Reid R, Woodcock S, Shimabukuro-Vornhagen A, Austen WG Jr, Kobzik L, Zhang M, Hechtman HB, Moore FD Jr, Carroll MC: Functional activity of natural antibody is altered in Cr2-deficient mice. Immunology
18. Zhang M, Alicot EM, Chiu I, Li J, Verna N, Vorup-Jensen T, Kessler B, Shimaoka M, Chan R, Friend D, et al.: Identification of the target self-antigens in reperfusion injury. J Exp Med
19. Chan RK, Verna N, Afnan J, Zhang M, Ibrahim S, Carroll MC, Moore FD Jr: Attenuation of skeletal muscle reperfusion injury with intravenous 12 amino acid peptides that bind to pathogenic IgM. Surgery
20. Suber F, Carroll MC, Moore FD Jr: Innate response to self-antigen significantly exacerbates burn wound depth. Proc Natl Acad Sci U S A
21. Ibrahim SI, Chan RK, Ding Y, Verna N, Suber F, Oakes SM, Hechtman HB, Moore FD Jr: Survival during murine hemorrhagic shock is improved in the absence of antibodies. J Am Coll Surg
22. Schur P: Complement studies of sera and other biological fluids. Hum Pathol
23. Patterson CE, Rhoades RA, Garcia JGN: Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung. J Appl Physiol
24. Hill J, Lindsay TF, Ortiz F, Yeh CG, Hechtman HB, Moore FD Jr: Soluble complement receptor type 1 ameliorates the local and remote organ injury following intestinal ischemia-reperfusion in the rat. J Immunol
25. Austen WG Jr, Kobzik L, Carroll MC, Hechtman HB, Moore FD Jr: The role of complement and natural antibody in intestinal ischemia-reperfusion injury. Int J Immunopathol Pharmacol
26. Solomkim JS, Nelson RD, Chenoweth DE, Solem LD, Simmons RL: Regulation of neutrophil migratory function in burn injury by complement activation products. Ann Surg
27. Moore FD Jr, Davis C, Rodrick M, Mannick JA, Fearon DT: Neutrophil activation in thermal injury as assessed by complement receptor upregulation. N Engl J Med
28. Chan RK, Ding G, Verna N, Ibrahim SI, Oakes S, Hechtman HB, Moore FD Jr: IgM binding to injured tissues precedes complement activation during skeletal muscle ischemia-reperfusion. J Surg Res
29. Fleming SD, Sh ea-Donohue T, Guthridge JM, Kulik L, Waldschmidt TJ, Gipson MG, Tsokos GC, Holers VM: Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire. J Immunol
30. Links Menezes J, Hierholzer C, Watkins SC, Lyons V, Peitzman AB, Billiar TR, Tweardy DJ, Harbrecht BG: A novel nitric oxide scavenger decreases liver injury and improves survival after hemorrhagic shock. Am J Physiol
31. Akabori H, Yamamoto H, Tsuchihashi H, Mori T, Fujino K, Shimizu T, Endo Y, Tani T: Transient receptor potential vanilloid 1 antagonist, capsazepine, improves survival in a rat hemorrhagic shock model. Ann Surg
32. Zhang M, Michael LH, Grosjean SA, Kelly RA, Carroll MC, Entman ML: The role of natural IgM in myocardial ischemia-reperfusion injury. J Mol Cell Cardiol
33. Chan RK, Austen WG Jr, Ibrahim S, Ding GY, Verna N, Hechtman H, Moore FD Jr: Reperfusion injury to skeletal muscle affects primarily type II muscle fibers. J Surg Res
IgM: Immunoglobulin M, a 750,000-kd pentameric antibody with 10 antigen-binding sites. Serum concentration 0.5 to 1 mg/mL.
Natural IgM: A subclass of IgM produced by B1 lymphocytes. The antigen specificity is gene encoded present at birth, and innate to each individual and species.
IgG: Immunoglobulin G, a 150,000-kd antibody with 2 antigen-binding sites. Antigen specificity and affinity increase with exposure to antigen through a multistep multicellular process. Produced by B2 lymphocytes of lymph nodes, spleen, and other organs.
B1 lymphocytes: Responsible for natural IgM production. CD5+ lymphocytes, identifiable by cell surface antigens surface IgM and CD11b. Primary population is self-replicating and resides in body cavities.
C3: Major complement protein involved in all three activation pathways.
C3b: C3 fragment that covalently binds to complement activators. Serves as scaffold for formation of lytic complement complex and amplified C3 cleavage. Serves as receptive antigen for immunological cell complement receptors. Stable.
C4b: Fragment of classical complement pathway C4 protein that binds to complement activators. Serves as scaffold for formation of C3-cleaving enzymes. Labile.
C1q: Collagenous protein that interacts with IgG or IgM to produce activation of the classical complement pathway.
C5b-9: Complement cell lytic structure that inserts into complement-activating membranes.
sCR1: Soluble form of complement receptor 1, the C3b receptor of immunological cells. Has potent complement inhibitory activity.
N2: a 12-amino acid peptide that is identical to the highly conserved hinge region of nonmuscle myosin heavy chain IIC: LMKNMDPLNDNV. Previously shown to prevent reperfusion injury.
SMM: a 12-amino acid peptide that is identical to the hinge region of skeletal muscle myosin heavy chain: LDKNKDPLNETV. Used as a control in these experiments.