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Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: in vivo iNOS expression pattern in (septic) shock

Jennyfer Bultinck^*, Patrick Sips^*, Luc Vakaet, Peter Brouckaert^* and Anje Cauwels^*,1

^* Department for Molecular Biomedical Research, Ghent University and VIB, Ghent, Belgium; and

Department of Radiotherapy, Ghent University Hospital, Ghent, Belgium

¹Correspondence: Department for Molecular Biomedical Research, Ghent University/VIB, Technologiepark 927, Ghent 9052, Belgium. E-mail: anje.cauwels{at}dmbr.ugent.be

SPECIFIC AIMS

Three different isoforms of NO synthase (NOS) exist: endothelial (e), neuronal (n), and inducible (i) NOS. As opposed to the constitutive enzymes eNOS and nNOS, iNOS is synthesized de novo during inflammation and produces large amounts of NO over prolonged periods of time. Septic shock is the leading cause of death in noncoronary intensive care units. Lipopolysaccharide (LPS), a constituent of the outer membrane of Gram-negative bacteria, mimics most of the septic effects and is widely used as an experimental model for septic shock. NO plays a controversial role in the pathophysiology of (septic) shock. Since it causes hypotension and tissue damage, NO may be considered detrimental to the host. However, NO may also confer protection to the host as an anti-inflammatory, antioxidant, and/or anti-apoptotic agent. This ambiguity may relate to its isoform-specific production but also to differences in cellular and/or temporal iNOS expression.

The iNOS enzyme was originally identified in macrophages. Inflammatory stimuli may induce the production of (iNOS-derived) NO in several hematopoietic cell lines, such as macrophages, neutrophils, eosinophils and lymphocytes. Hence, and because of their well-known role in inflammatory diseases such as septic shock, immune cells have generally been considered the principal in vivo source of systemic high-output NO. To verify this, we performed bone marrow transplantation (BMT) experiments between wild-type (WT) and iNOS- or TNF-R1-deficient mice and challenged these chimeric mice with shock-inducing TNF, LPS, or live Salmonella. In comparison, we also infected mice with BCG, which causes a granulomatous inflammatory reaction. In addition, the in vivo expression pattern of iNOS mRNA and protein was investigated.

PRINCIPAL FINDINGS

1. Parenchymal cells are the only source of systemic NO_x^– in TNF- or LPS-induced shock and in a bacteremic model of septic shock
To investigate the involvement of hematopoietic cells in systemic NO_x^– production after TNF or LPS, irradiated iNOS^–/– mice were reconstituted with WT BM cells and vice versa. As a control, WT and iNOS^–/– mice were, after irradiation, transplanted with homologous BM cells. To check the BMT effectiveness, the genotype of circulating WBCs was determined. As shown in Fig. 1 (insert), virtually all the hematopoietic cells of the reconstituted mice were of the donor genotype. Three days later, the mice were intravenously injected with lethal doses of TNF, E. coli, or S. abortus equi LPS. Systemic interleukin (IL)-6 levels 8 h after challenge were comparable (not shown). However, TNF or LPS did not induce any detectable NO_x^– in circulation of iNOS^–/– mice with an iNOS^+/+ hematopoietic cell population, while they clearly did in mice that express iNOS in every cell but the hematopoietic cells (Fig. 1A ). Therefore, in TNF- or LPS-challenged mice parenchymal cells are fully responsible for the systemic NO_x^– production. For TNF, we confirmed this result with a BMT between WT and TNF-R1^–/– mice (not shown).

To substantiate the insignificance of immune cells in NO_x^– production during shock, we set up an infectious Salmonella enteritidis model. As reported before, iNOS^–/– mice were more susceptible to Salmonella infection (not shown). Between 18 to 30 h after infection, significant amounts of circulating NO_x^– were present in Salmonella-infected WT to WT mice, as well as in WT mice with iNOS^–/– BM. In contrast, however, only very low and insignificant levels of NO_x^– were found in chimeric mice with iNOS-negative parenchymal cells (not shown), indicating that in a bacteremic model of septic shock there is also a predominant role for parenchymal cells in systemic NO_x^– production.

2. Hematopoietic cells are responsible for systemic NO_x^– production during granulomatous inflammation
In an analogous BMT experiment between WT and iNOS^–/– mice, we found that hematopoietic cells are solely responsible for systemic NO_x^– production during BCG infection. Only in mice with a WT parenchymal compartment, an extra TNF challenge caused an additional increase in NO_x^– production (not shown).

3. Expression patterns of iNOS
To elucidate the induction pattern of iNOS, we first determined the iNOS mRNA and protein levels in various organs via real time-quantitative polymerase chain reaction (PCR) and WB analysis, respectively. After BCG infection, iNOS was strongly detected in liver, lung, and spleen and weakly in kidney and heart, corresponding with the organs in which granulomas could be detected (not shown). After TNF or LPS, iNOS was strongly detected in liver, kidney, jejunum, and colon. To examine the cellular expression pattern of iNOS, we performed immunohistochemistry (IHC). Identical iNOS expression patterns were observed after TNF or LPS (Fig. 2 A–H), although less pronounced after LPS, correlating with the systemic NO_x^– levels. Positive iNOS staining was detected in hepatocytes, often specifically at the canalicular membrane (Fig. 2A, E ). In the kidney, epithelial cells of pelvis (Fig. 2B, F ) and tubuli (not shown) stained positive. Throughout the gastrointestinal tract, apical epithelial cells clearly expressed iNOS (Fig. 2C, G ), as well as the paneth cells of the jejunum (Fig. 2D, H ). In addition, after LPS we also observed some iNOS-positive Kupffer cells and occasional granulocytes and/or monocytes in liver, lung, spleen, kidney and heart (not shown). Nevertheless, despite their ability to express iNOS protein in vivo, our BMT experiments indicate that these WBCs do not contribute significantly to the massive systemic NO_x^– production.

One day after the inoculation of Salmonella enteritidis, similar iNOS expression patterns were detected as for LPS (Fig. 2I-L ). Salmonella infection also occasionally induced some small iNOS-positive granulomas (not shown).

BCG infection induces the formation of larger granulomas, particularly in liver, lung, and spleen, and occasionally in kidney and heart. IHC revealed iNOS expression exclusively in the epithelioid macrophages within those granulomas (Fig. 2M-P ).

CONCLUSIONS AND SIGNIFICANCE

For a long time, it has been accepted that leukocytes are the main source of systemic NO in infectious and inflammatory diseases, including septic shock. However, we here provide conclusive in vivo evidence to refute this general assumption. Our study clearly establishes a predominant role for parenchymal cells in systemic NO_x^– production during endotoxic and bacteremic (septic) shock. We also identified iNOS-expressing hepatocytes, paneth cells, and epithelial cells of the gut and kidney as the parenchymal source of systemic NO after TNF, LPS, or Salmonella infection. Since liver and intestinal tract represent two of the largest organs within the mammalian body, they correspond with a vast amount of iNOS-expressing cells. iNOS-expressing hepatocytes (including the specific canalicular staining), Kupffer cells, and gastrointestinal epithelial cells have been described before. Interestingly, we also found distinctive iNOS expression in paneth cells, which has never been reported before. Paneth cells release several antimicrobial peptides and proteins and thus have an important role in innate mucosal immunity. The well-defined function of iNOS-derived NO in innate immunity makes iNOS induction in paneth cells very plausible.

As evidenced by our immunohistochemical study and that of others, LPS treatment of rodents induces the expression of iNOS in monocytes, macrophages, and granulocytes. However, and surprisingly, those cells do not contribute significantly to systemic NO production, as taught by our BMT experiments. Several possible explanations may be envisioned. It could be that those few cells are simply insufficient to contribute significantly. Other explanations could be that the NO is locally captured in a rapid reaction like that with other radicals or that iNOS expression in these WBCs does not correlate with NO production. Indeed, when L-arg is limited, for example, iNOS produces superoxide rather than NO in a so-called "uncoupled" reaction.

Whereas parenchymal cells are the major source of systemic NO during TNF-, LPS-, or Salmonella-induced shock, hematopoietic cells (more specifically the granulomar epithelioid macrophages) represent the main source of systemic NO during BCG infection.

In conclusion, our in vivo study reveals a differential role for hematopoietic cells in systemic NO production. It may be interesting to add that the generation of induced NO by human monocytes/macrophages has long been a subject of great controversy. Although humans produce significant quantities of NO during infection or inflammation, in vitro treatment of human monocytes/macrophages failed to elicit NO release, in contrast to murine macrophages. It has always been believed that this discrepancy could be attributed to an inherent difference between mice and men. Our data indicate that differences between in vitro and in vivo observations, rather than species diversities, are responsible for this discrepancy and that in both species the role of monocytes/macrophages as the source of shock-inducing NO is much less prominent than generally assumed.

View larger version (31K): [in this window] [in a new window]   Figure 1. Serum NOx– levels in TNF- or LPS-treated WT, iNOS–/–, and BMT mice. Mice were injected iv with PBS, 12.5 µg TNF, 300 µg E. coli LPS, or 150 µg S. abortus equi LPS. Serum NOx– levels were determined 8 h later. ***P < 0.001, as compared with corresponding treatment in the WT to WT group. Plotted are means ± SD; numbers above bars are number of mice. Insert: PCR reactions, specific for the WT or deficient iNOS allele, were performed on WBC DNA of BMT mice. I: WT to WT, II: iNOS–/– to WT, III: WT to iNOS–/–, IV: iNOS–/– to iNOS–/–, M: marker.

View larger version (113K): [in this window] [in a new window]   Figure 2. iNOS-specific IHC. Organs were collected 8 h after 5 µg TNF or 150 µg S. abortus equi LPS, 24 h after 800 cfu of S. enteritidis, or 2 h after PBS in BCG-infected mice. Arrows in A indicate canalicular membranes of hepatocytes. Magnification = x40 (J, N), x60 (A, C, E, G), or x100 (B, D, F, H, I, K, L, M, O, P).

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic presentation of in vivo cellular source of iNOS-derived NO in various inflammatory settings. BM transplantations between WT and iNOS–/– mice revealed that hematopoietic cells are fully responsible for systemic NO production during BCG infection. However, after TNF, LPS or Salmonella treatment, most, if not all, systemic NO originates from non-hematopoietic cells, which is in contrast with the general assumption, based on in vitro studies, that macrophages are the principle source of iNOS-derived NO during inflammatory shock.

FOOTNOTES

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