[Host inflammatory and anti-inflammatory response during sepsis]. / Réponse inflammatoire et anti-inflammatoire de l'hôte au cours du sepsis.

Sepsis still remains the major complication for patients admitted in intensive care units (ICU), and is responsible for numerous deaths. ICU patients admitted after sepsis, hemorrhagic shock, severe trauma, severe burns or major surgery show a systemic inflammatory response syndrome (SIRS). This syndrome is characterized by an exacerbation of inflammation, with increased levels of pro- (IL-1ß, TNFα, IL-6, IL-8) as well as anti-inflammatory (IL-10, IL-1Ra, TGFß) cytokines into their bloodstream. During sepsis, the bacteria release microbial motifs such as peptidoglycan, lipopolysaccharide (LPS) and DNA that initiate the inflammatory response, and are involved in the onset of multiple organ failure. The same microbial motifs can also be found in patients with a SIRS of non-infectious origin, following the translocation of bacteria from their digestive tract. This translocation is certainly contributing to the difficulty of discriminating between septic and SIRS patients using biological markers. Furthermore, the host response is accompanied by an alteration of the ex vivo response of circulating leukocytes, particularly monocytes. This hyporesponsiveness to LPS is associated with a decreased activation of the transcription factor NF-κB (required for the expression of pro-inflammatory cytokines) and an increased expression of negative regulators of the NF-κB pathway. However, the leukocyte hyporesponsiveness is not a global phenomenon, it depends on the type of patient, on the receptor-activator pair, on the timing, and on the cytokine.

Reprogramming of circulatory cells in sepsis and SIRS.

Immune status is altered in patients with sepsis or non-infectious systemic inflammatory response syndrome (SIRS). Reduced ex-vivo TNF production by endotoxin-activated monocytes has been regularly reported. This observation is reminiscent of the phenomenon of endotoxin tolerance, and the term 'leukocyte reprogramming' well defines this phenomenon. This review will outline that the hyporesponsiveness of circulating leukocytes is not a generalized phenomenon in sepsis and SIRS. Indeed, the nature of the insult (i.e. infectious versus non-infectious SIRS; under anesthesia [surgery] or not [trauma, burn]), the nature of the activator used to trigger leukocytes (i.e. different Toll-like receptor ligands or whole bacteria), the nature of the cell culture (i.e. isolated monocytes versus peripheral blood mononuclear cells versus whole blood assays), and the nature of the analyzed cytokines (e.g. IL-1beta versus IL-1ra; TNF versus IL-10) have a profound influence on the outcome of the response.

Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis.

UNLABELLED: Reduced mitochondrial membrane potential (Delta(Psi)m), which is considered as an initial and irreversible step towards apoptosis, as well as cell death regulating proteins, such as Fas, Hsp70, or Bcl-2, may play an important role in sepsis. We studied the relationship between sepsis severity and peripheral blood monocyte Delta(Psi)m, cell death (necrosis and apoptosis), soluble Fas ligand, Hsp70, and Bcl-2 expression over time in 18 patients with sepsis, and compared these data with those of a group of 17 healthy control subjects. All measurements were performed within 3 d of the onset of severe sepsis (T1), then 7 to 10 d later (T2), and finally at hospital discharge (T3). Delta(Psi)m was expressed as the percent monocytes with altered Delta(Psi)m (%Delta(Psi)m). Patients with sepsis had greater %Delta(Psi)m at T1 and T2 but not at T3 (14.6 +/- 2.6% and 15.9 +/- 2%, respectively, versus control 6.6 +/- 0.2%, p < 0.01). Septic patients exhibited greater cell death in their monocytes and had greater Hsp70 expression only at T1. Bcl-2 levels were similar in septic and control subjects. Comparing survivors with non-survivors of sepsis, nonsurvivors had a greater %Delta(Psi)m at T1 (26.4 +/- 5.3% versus 10.1 +/- 2.7%, p < 0.01) and a significant decrease in Bcl-2 expression, whereas no difference was found in Hsp70 levels. These results indicate that mitochondrial dysfunction and subsequent cell death occur in severe sepsis and suggest that %Delta(Psi)m is a marker of severity in human sepsis. KEYWORDS: mitochondria; apoptosis; sepsis; heat-shock protein 70; proto-oncogene protein c-Bcl-2

Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review.

Sepsis and non-infectious systemic inflammatory response syndrome (SIRS) are paradoxically associated with an exacerbated production of cytokines, as assessed by their presence in biological fluids, and a diminished ability of circulating leukocytes to produce cytokine upon in vitro activation. In this review, we depict that the observed cellular hyporeactivity is not a global phenomenon and that some signalling pathways are unaltered and allow the cells to respond normally to certain stimuli. Furthermore, we illustrate that during sepsis and SIRS, cells derived from tissues are either fully responsive to ex vivo stimuli or even primed, in contrast to cells derived from hematopoietic compartments (blood, spleen, etc.) which are hyporeactive. In addition to cytokine production, nuclear factor-kappa B (NF-kappa B) status within leukocytes can be used as a useful marker of hypo- or hyper-reactivity. We illustrate that the immune-depression reported in sepsis and SIRS patients, often revealed by a diminished capacity of leukocytes to respond to lipopolysaccharide, is not a generalized phenomenon and that SIRS is associated with a compartmentalized responsiveness which involves either anergic or primed cells.

Long-term-impaired expression of nuclear factor-kappa B and I kappa B alpha in peripheral blood mononuclear cells of trauma patients.

Nuclear factor (NF)-kappa B expression and dimer characteristics were studied in peripheral blood mononuclear cells (PBMCs) of major-trauma patients and healthy controls. Analysis of PBMCs on days 1, 3, 5, and 10 after trauma revealed that expression of both p65p50 heterodimers and p50p50 homodimers was significantly reduced compared with that in controls. In vitro lipopolysaccharide (LPS) stimulation of PBMCs induced NF-kappa B translocation. However, throughout the survey, p65p50 activation remained significantly lower in trauma patients than in controls. After LPS stimulation in vitro, the p65p50/p50p50 ratio was significantly lower in PBMCs from trauma patients than from healthy controls. The ex vivo expression of I kappa B alpha was higher in PBMCs of controls than of trauma patients. LPS did not induce I kappa B expression in PBMCs from trauma patients, but strong induction was obtained with staphylococci, suggesting that this defect is not universal and depends on the nature of the activating signal. Although no direct correlation was found between levels of interleukin-10 or transforming growth factor-beta and NF-kappa B, these immunosuppressive cytokines were significantly elevated in trauma patients by 10 days after admission. The long-term low-basal and LPS-induced nuclear translocation of NF-kappa B recalled long-term immunoparalysis observed in patients with severe inflammatory stress such as trauma.

NF-kappaB expression in mononuclear cells of patients with sepsis resembles that observed in lipopolysaccharide tolerance.

The expression of NF-kappaB was studied in freshly isolated peripheral blood mononuclear cells (PBMC) of patients with severe sepsis and major trauma. The expression of p65p50 heterodimer, the active form of NF-kappaB, was significantly reduced for all patients as compared with control subjects. The p50p50 homodimer, an inhibitory form of NF-kappaB, was reduced in the survivors of sepsis and in patients with trauma. Subsequent in vitro stimulation of PBMC with lipopolysaccharide (LPS) did not induce further NF-kappaB nuclear translocation: the survivors of sepsis and trauma patients showed low expression of both p65p50 and p50p50, whereas nonsurvivors of sepsis showed a predominance of the inactive homodimer and a low p65p50/p50p50 ratio when compared with control subjects. In the later group of patients there was a reverse correlation between plasma IL-10 levels and the p65p50/p50p50 ratio after in vitro LPS stimulation (r = -0.8, p = 0.04). The reduced expression of nuclear NF-kappaB was not due to its inhibition by IkappaBalpha, as very low expression of IkappaBalpha, as well as low levels of p65 and p50 were found in the cytoplasm of PBMC from patients with sepsis and trauma when compared with control subjects. These results demonstrate that upon LPS activation, PBMC of patients with systemic inflammatory response syndrome show patterns of NF-kappaB expression that resemble those reported during LPS tolerance: global down-regulation of NF-kappaB in survivors of sepsis and trauma patients and the presence of large amounts of the inactive homodimer in the nonsurvivors of sepsis.

Paradoxical priming effects of IL-10 on cytokine production.

IL-10 is a well-known immunosuppressive and/or anti-inflammatory cytokine. However, we report in vitro experimental studies in which IL-10 primed leukocytes and led to an enhanced production of tumor necrosis factor (TNF) upon further stimulation by lipopolysaccharide (LPS). Monocytes and peripheral blood mononuclear cells (PBMC) prepared from whole blood maintained for 20 h at 37 degrees C in the presence of recombinant human IL-10 had an enhanced capacity to produce TNF in response to LPS. In addition to TNF, LPS-induced IL-6 and spontaneous IL-1ra production were also enhanced. When isolated PBMC were first cultured for 20 h in the presence of IL-10 on Teflon to prevent adherence, washed to remove IL-10 and then further cultured in plastic dishes for an additional 20 h in the presence of LPS or IL-1beta, an enhanced release of TNF was observed. This was not the case when PBMC were pre-cultured in plastic multidishes in the presence of IL-10. TNF mRNA expression induced by LPS was decreased when the pre-treatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when cells were pre-cultured with IL-10 on Teflon. Furthermore, NFkappaB translocation following LPS activation was higher after IL-10 pre-treatment on Teflon than on plastic. Interestingly, an enhanced frequency of CD16 and CD68(+) cells among the CD14(+) cells was observed in the presence of IL-10, independently of the pre-culture conditions of the PBMC. Altogether, these results indicate that the IL-10-induced up-regulation of cytokine production depends on the prevention of monocyte adherence by red cells in the whole blood assays or by cultures of PBMC on Teflon. In contrast, the adherence parameter has no effect on the IL-10-induced modulation of some monocyte surface markers.

Effect of amino acid substitutions in the heavy chain CDR3 of an autoantibody on its reactivity.

In this study, we applied site-directed mutagenesis to the Fab fragment of a mouse IgM (IE12) that was previously shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. Its native structure was very similar to that of a polyreactive natural IgM (ppc15-30). Indeed, they both use the same light chain and the same VH, D and JH segments. However, the N regions differ and the D is translated in two different reading frames, giving different amino acid compositions of the heavy chain CDR3 (HCDR3). Site-directed mutagenesis modified the HCDR3 of IE12 compared to that of the natural antibody and the resulting effects on its reactivity were analyzed. Because the HCDR3 of IE12 is very rich in aliphatic residues, which are hydrophobic, we replaced them with the more hydrophilic residues of the HCDR3 of the polyreactive IgM. In addition, we evaluated the impact of the proline residues in the HCDR3 of IE12 on its activity, because they are known to restrict backbone flexibility. We found that a more hydrophilic HCDR3 conferred to the IE12 Fab a polyreactive profile. Prolines seem to play an important role in this context, because when they were replaced by glycines, the resulting Fab fragments were highly polyreactive. Our results suggest that, for polyreactivity, hydrophilicity and a certain plasticity of the HCDR3 seem to be necessary. Greater flexibility of the CDR, particularly the HCDR3, might be an important characteristic for polyreactive antibodies.

Effect of temperature on the reactivities of polyreactive and monospecific monoclonal IgG antibodies.

In this study, the reactivities of two groups of murine monoclonal antibodies (mAbs) of the IgG isotype were compared by ELISA at various temperatures (Ts) (range: 4-56 degrees C). The first group was constituted of 4 polyreactive mAbs that reacted with various antigens (Ags), such as actin, myosin and tubulin. The second contained 3 commercially available monoreactive mAbs specific to actin, myosin or tubulin. The binding of the monospecific mAbs to their Ags was modified only slightly at the other Ts compared with binding at 37 degrees C. In contrast, the activities of the polyreactive IgGs were considerably modified depending upon the T during incubation on Ag. In a second series of experiments, the effects of the T of the washes and conjugate incubation on the mAb/Ag interaction were evaluated. In these experiments, all steps and incubations were carried out at 37 or 4 degrees C. The values were then compared to those obtained when the washes and conjugate incubation were performed at room temperature. These two protocols generated very little difference in terms of monospecific mAbs. For polyreactive IgG, the values were generally lower when the incubations were carried out at 4 degrees C. However, on the whole, the effect of the T of the washes and conjugate incubation was negligible when the mAb/Ag complex had already formed on the polystyrene plate. Furthermore, at 4 degrees C, 2 of the polyreactive mAbs behaved like monospecific antibodies, while the other 2 remained polyspecific. It can be concluded from these experiments that the reactivities of polyreactive mAbs are more T-sensitive than those of monoreactive mAbs. This seems to indicate that they may possess a more plastic structure and thus may more easily undergo deformation when subjected to non-physiological conditions. In addition, the fact that the polyreactive mAbs showed the same variations for the 3 Ag tested suggests that the same paratope could be involved in all reactions.

Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions.

In this report, we describe the expression system that enabled us to produce in Escherichia coli the Fab fragment of a mouse IgM that has previously been shown to inhibit the binding of IgG to autoantigens by interacting with their variable regions. In our system, both light chain and heavy chain fragments were put under the control of the malE promoter. The light chain was fused to the MalE signal sequence, while the heavy chain variable and first constant region were fused to the alkaline phosphatase signal sequence. In this system, after induction of the promoter with maltose, the Fab fragment could be detected in a periplasmic extract of the bacteria by Western blotting and also by ELISA. This Fab fragment was purified on a goat anti-mouse immunoglobulin immunoadsorbent and biotinylated. The Fab fragment produced by E.coli reacted with the trinitrophenyl (TNP) hapten and F(ab')2 fragments of mouse IgG and these reactivities could be specifically inhibited by the corresponding soluble antigens. The dissociation constants of this Fab were 1.65 x 10(-6) M for TNP and 5 x 10(-6) M for IgG F(ab')2 fragments, indicating that the affinity of the Fab fragment compared with that of the whole IgM molecule was similar for TNP but was lower for IgG F(ab')2 fragments.

Effects of pH or ionic strength on the reactivities of mono- and polyspecific IgG antibodies.

The reactivities of mono- and polyspecific mouse monoclonal antibodies (mAb) were compared by ELISA using immobilized antigens under different conditions, varying the pH or the NaCl concentration. The monospecific group was composed of 6 IgG directed against Staphylococcus aureus capsular polysaccharides and of 3 IgG specific to actin, myosin or tubulin. These antibodies were compared with 6 polyreactive mAb also of the IgG isotype. Marked differences were noted between the reactivities of the mono- and polyreactive IgG. pH variations had little or no effect on the reactivity of monospecific mAb to polysaccharides or to proteins. In contrast, the binding of polyreactive mAb was dependent on the pH, and the profile differed for each antigen. The NaCl concentration had opposite effects on mono- and polyreactive mAb: the binding of almost all the monoreactive mAb was increased at high NaCl concentration, while it was decreased for polyreactive mAb. In contrast, the effects of varying the pH or ionic strength on the coated antigens were negligible. The variation coefficients calculated for the pH and NaCl concentration were higher for the polyreactive mAb under study, which seems to indicate that electrostatic interactions and charged residues might be more important for these mAb than for the monoreactive ones. This characteristic might be one explanation for the particular properties of these polyreactive antibodies.

Reactivity and structure of a mouse anti-F(ab')2 IgM. Comparison of its variable region sequences with those of a structurally close polyreactive natural IgM.

IE12 is a monoclonal IgM with strong anti-F(ab')2 activity that inhibits the binding of normal mouse IgG to self antigens. In this study, we found that this IgM was also reactive with several monoclonal antibodies (mAbs), myeloma proteins and B lymphocytes from normal BALB/c mouse. The nucleotide sequences of the variable region of the heavy and light chains of IE12 were determined, and compared to those of another mAb already described in the literature. This mAb uses the same light chain and also the same VH, D and JH segments, but unlike IE12, is polyreactive. The comparison of the amino acid composition of these two mAbs and of the computer predictions for their structure and hydrophilicity indicated that the most striking difference between them was located in the third complementarity determining region (CDR3) of the heavy chain. Indeed, they used the same D segment but translated in two different reading frames, leading to different amino acid compositions. The CDR3 of IE12 contains aliphatic amino acids, while that of the polyreactive IgM does not. In addition, IE12 has two prolines, one at each at each extremity of its D segment, that could confer a certain rigidity to this region. Finally, the CDR3 of IE12 is predicted to be hydrophobic, while the one of the polyreactive IgM is predicted to be hydrophilic and more flexible, suggesting that the hydrophilicity and the flexibility of this region might be critical for polyreactivity.

Monoclonal IgG and IgM autoantibodies obtained after polyclonal activation, show reactivities similar to those of polyclonal natural autoantibodies.

In this study, we described the properties of two groups of polyreactive IgG monoclonal antibodies (mAb) derived from splenocytes of a Plasmodium chabaudi-infected BALB/c mouse. These IgG mAb reacted with self antigens, but not with the parasite. Depending upon the antigen under study, they showed low (10(-5) M) to high (10(-8) M) affinities. When examined by immunoblotting using mouse organ extracts as the antigen source, each IgG mAb had a different reactivity profile. The IgG2b, but not the IgG2a mAb group, reacted with F(ab')2 fragments of IgG from normal mouse sera, as well as with F(ab')2 fragments of other mAb from the same fusion. Their anti-F(ab')2 activity was inhibited by various antigens, suggesting that the binding sites for these antigens and F(ab')2 fragments were close or overlapping. We also characterized two monoclonal IgM that were strongly and almost exclusively reactive with polyclonal F(ab')2 fragments of normal IgG, and that inhibited the binding of normal polyclonal IgG to self antigens. We previously described such properties for polyclonal IgM from normal mouse sera. The binding of the F(ab')2-reactive IgG mAb to self antigens was also inhibited by normal polyclonal IgM. These results indicate that the IgG and IgM obtained after polyclonal stimulation, exhibit characteristics similar to those of autoantibodies present in normal mouse sera. Furthermore, they confirm our previous studies showing the polyreactivity of polyclonal IgG. Finally, they also show that such IgG mAb exhibit properties similar to those of natural IgM, namely polyreactivity, affinity and connectivity.