Proinflammatory kupffer cell alterations after femur fracture trauma and sepsis in rats.

This study examined effects of trauma and sepsis on Kupffer cell function. When CBA/J mice had femur fracture (FFx), no deaths occurred. After cecal ligation and puncture (CLP), 44% died. Following combined injuries (FFx + CLP), mortality increased to 60%, suggesting a deleterious effect between FFx + CLP. Kupffer cell ablation with GdCI3 decreased mortality to 13% after CLP and 5% after FFx + CLP. After FFx, CLP, and FFx + CLP, Kupffer cells isolated from Sprague-Dawley rats produced 720%, 1,100%, and 2,130% more O2. than sham, respectively. Phagocytosis increased 320%, 610%, and 150%. Kupffer cell PGE2 production also increased 300%, 510%, and 300% over sham. After FFx alone, TNF-alpha production decreased 40%. By contrast, CLP and FFx + CLP increased TNF-alpha release 25% and 100%, respectively. After FFx, NO. production decreased 44%, whereas NO increased 280% and 260% after CLP and FFx + CLP. These findings indicate that Kupffer cells mediate mortality after CLP and FFx + CLP. Increased mortality is associated with a more proinflammatory and less antimicrobial Kupffer cell phenotype.

Host priming, not target antigen type, decides rejection rate in mice primed with MHC II "knockout" cultured keratinocytes.

BACKGROUND: Lack of skin for autograft continues to be problematic in patients with large burns. Allograft and xenograft have been used, but are prone to rapid rejection. Use of cultured keratinocytes (CK) and major histocompatibility complex (MHC) II "knockout" grafts leads to prolonged graft survival compared to allograft. Whether this prolongation is secondary to decreased priming efficacy or target recognition is unknown. Whether a combination of these techniques would generate a less immunogenic allograft remains to be determined. METHODS: CBA mice (n = 100) were flank-grafted with full thickness C57BL/6 (B6 FT), B6 cultured keratinocytes (B6 CK), B6 major histocompatibility complex II "knockout" full thickness (KO II FT), B6 major histocompatibility complex II "knockout" cultured keratinocytes (KO II CK), or a full thickness autograft (Auto). Three weeks after priming flank grafting, B6, MHC I (KO I), and KO II full thickness tail grafts were placed on each mouse. Tail graft rejection was assessed daily by an observer blinded to flank and tail-graft type. A 4-point grading system for graft color, hair loss, and texture was used. RESULTS: Animals primed with KO II CK flank grafts had increased survival of tail grafts over B6 FT flank grafted controls (12.3 +/- 1.05 vs 10.1 +/- 1.00, P < 0.05). Within flank graft groups, however, B6, KO I, and KO II tail graft survival was similar. CONCLUSIONS: KO II CK allografts decrease host priming compared to normal B6 FT allograft. MHC deletion (KO I or KO II) does not protect a target graft from rejection in a primed host. CK and KO techniques may offer a less immunogenic allograft and a readily available source of wound coverage in patients with extensive burns.

The effector component of the cytotoxic T-lymphocyte response has a biphasic pattern after burn injury.

INTRODUCTION: Burn injury delays allograft rejection and impairs the host defense against infection. These functions are mediated via the cytotoxic T-lymphocyte (CTL) response. The CTL response is divided into antigen recognition/processing and effector phases. Presensitization allows selective analysis of changes, induced by burn injury, in the effector limb of the CTL response in relation to time and burn size. METHODS: Anesthetized CBA mice were primed with either a flank allograft from C57BL/6 (B6) mice or an autograft (negative control). Five weeks after grafting, animals were anesthetized and received either a 0, 20, or 40% burn. Spleens were harvested 3, 7, 10, and 14 days after burn injury (n = 96), cocultured with B6 stimulator splenocytes, and assessed for CTL response to radiolabeled allogeneic targets in a 51Cr release assay. In experiment 2, spleens were harvested from unburned and 40% burned animals on Postburn Days 3 and 14. After triple staining, cells were analyzed by flow cytometry for CD4, CD8, and CD25 antigens. In experiment 3, splenocytes from 0 and 40% burned animals on Postburn Days 3 and 14, were cocultured with B6 stimulators for 5 days. Supernatants were evaluated for interleukin (IL)-2, IL-5, and interferon-gamma (IFN-gamma) using ELISA: RESULTS: The CTL response for 20 and 40% burned animals decreased 3 days postburn (-11.9 and -30.1%, P < 0.05), returned to baseline in 7-10 days, and was increased by 14 days postburn (15.8 and 22.6%, P < 0.05). The T-helper lymphocyte population (CD4) from 40% burn animals was significantly decreased on Postburn Days 3 and 14 (10.12 +/- 0.45% vs 11.78 +/- 0.29% and 10.19 +/- 0.24% vs 14.21 +/- 0.97%, respectively, P < 0.05). The CTL effector (CD8) splenocyte population was significantly higher in the burned animals on Postburn Day 14 (4.55% vs 3.71%, P < 0.05). On Postburn Day 3, average IL-5 production was higher in the burned animals (1.80 pg/ml vs 0.59 pg/ml, respectively, P < 0.05). The burn group, on Postburn Days 3 and 14, showed a decrease in mean IL-2 production (212.81 pg/ml vs 263.6 pg/ml and 342.7 pg/ml vs 421.4 pg/ml, respectively, P < 0.05). Mean IFN-gamma production on Postburn Days 3 and 14 was decreased in burned mice (263.75 pg/ml vs 285.57 pg/ml and 218.16 pg/ml vs 263.42 pg/ml, P < 0.05). CONCLUSIONS: Burn injury impairs the effector limb of the CTL response as a function of burn size in the immediate postburn period. CTL activity returns to baseline within 7-10 days postburn and has a rebound increase by Day 14. Early CTL suppression, after burn injury, may be due to a decrease in the T-helper subpopulation. The late increase in cytotoxicity may be secondary to an increase in the effector CTL population in the late postburn period. Burn injury causes a T-helper-2 phenotype as demonstrated by depressed IL-2 and IFN-gamma production and increased IL-5 production.

Adaptive Kupffer cell alterations after femur fracture trauma in rats.

Because Kupffer cells constitute the largest fixed macrophage population and reside at a strategic position in hepatic sinusoids, interacting with hepatocytes, circulating cells, and mediators from the gut, they may be important in the inflammatory response after injury. This study examined the effect of remote tissue injury on Kupffer cell function. Femurs of Sprague-Dawley rats were fractured under anesthesia. Subsequently, their livers were perfused for measurement of oxygen consumption and the isolation and culture of Kupffer cells. At 2 and 48 h after femur fracture, hepatic oxygen consumption increased 17 and 19%, respectively. Gadolinium chloride pretreatment to ablate Kupffer cells blocked this increase of hepatic oxygen consumption after femur fracture but had no effect in sham-operated animals. In Kupffer cells isolated and cultured 2 h after femur fracture, superoxide formation stimulated by phorbol ester increased eightfold, phagocytosis increased fourfold, and lipopolysaccharide (LPS)-stimulated prostaglandin E2 increased sixfold in comparison to sham-operated controls. In contrast, LPS-stimulated tumor necrosis factor-alpha and nitric oxide production decreased 50 and 60%, respectively. These data show that peripheral trauma rapidly induces changes in hepatic macrophages characterized by adaptation to a more antimicrobial and less proinflammatory phenotype.

An in vitro investigation into the effects of repetitive motion and nonsteroidal antiinflammatory medication on human tendon fibroblasts.

Soft tissue injuries due to repetitive motion are common sports injuries and are often treated with antiinflammatory therapies. We investigated the in vitro effects of repetitive motion and nonsteroidal antiinflammatory medication on human tendon fibroblasts. In addition, we studied the effects related to the presence of inflammatory cells. Repetitive motion was associated with an increased release of prostaglandin E2 and increased deoxyribonucleic acid (DNA) and protein synthesis. The presence of nonsteroidal antiinflammatory medication decreased prostaglandin E2 release and DNA synthesis but increased protein synthesis. Contact with macrophages caused a marked additional increase in prostaglandin E2 and a concomitant increase in DNA synthesis. Release of interleukin-6 by the macrophages also suggested that this cytokine plays a role in the response to repetitive motion. Our results can aid in the search for a more scientific approach to the treatment of soft tissue injuries associated with repetitive motion. They suggest that nonsteroidal antiinflammatory medication may have potentially negative effects during the proliferative phase of a healing since it was associated with decreased DNA synthesis. However, it may be beneficial in the maturation and remodeling phase since it stimulated protein synthesis.

The N haplotype of the murine beta-glucuronidase gene is altered in both its systemic regulation and its response to androgen induction.

A new haplotype of the beta-glucuronidase gene complex, [Gus]N, has been characterized following its transfer from the PAC/Cr strain to the standard strain C57BL/6J. The N haplotype contains a novel structural gene allele which encodes an allozyme differing from all previously characterized allozymes in both size and charge. Altered systemic regulation is exhibited by the [Gus]N haplotype. Multiple tissues contain levels of GUS protein that are 60 +/- 15% those found in the standard B haplotype. The regulatory mechanism for reduction is complex, involving tissue-specific changes in both enzyme synthesis and enzyme turnover. The changes in GUS protein synthesis do not result from changes in GUS mRNA levels. Instead, the amount of mature enzyme formed per mRNA molecule, or translational yield, is altered. These regulatory changes parallel those seen in other systemic regulatory variants of GUS which are also altered in translational yield. A commonality of mechanism among systemic regulatory variants of this gene is suggested. The N haplotype is also exceptional in the nature of its response to androgenic induction in kidney proximal tubule epithelial cells. The time course for GUS induction consists of a lag period followed by a progressive increase in mRNA, rate of enzyme synthesis, and enzyme activity. For the [Gus]N haplotype the lag is of an exceptionally short duration and the plateau is of a greater magnitude than for any haplotype previously described.

Androgen induction of beta-glucuronidase translational yield in submaxillary gland of B6.N mice.

Androgens induce the synthesis of murine beta-glucuronidase (GUS) 10-fold in the submaxillary gland of B6.PAC-Gusn mice without a concomitant increase in GUS mRNA levels. Since the rate of GUS synthesis per mRNA molecule, or translational yield, is a function of both the efficiency with which the message is translated and the fraction of newly made polypeptides that are incorporated into GUS tetramers, we conclude that androgen induces at least one of these components in the submaxillary gland of B6.N mice. Genetic variation in the submaxillary gland induction response was tested for using six congenic mice strains, each carrying a different haplotype of the Gus gene complex on a C57BL/6J genetic background. The results indicate that the DNA sequences determining androgen responsiveness of the Gus gene in the submaxillary gland are linked to the Gus gene complex and that the DNA sequences determining the submaxillary gland response are distinct from those determining the androgen induction of GUS mRNA in kidney.

Changes in translational yield regulate tissue-specific expression of beta-glucuronidase.

The number of beta-glucuronidase (GUS; beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) molecules per cell varies as much as 12-fold among mouse tissues. To identify the regulatory mechanisms responsible, estimates of the rates of GUS protein synthesis (ks) and degradation (kd) were obtained for six tissues in the B6.PAC-Gusn mouse strain, which carries the N haplotype of the GUS gene. Differences in enzyme levels among tissues were predominantly due to differences in rates of enzyme synthesis; only brain differed significantly in the rate of protein degradation. Typically, tissues contain about 2 molecules of GUS mRNA per cell. Differences in GUS mRNA levels were found among tissues, but these were not sufficient to account for observed differences in ks. This suggests that tissues differ in translational yield, which is defined as the product of the efficiency with which the GUS message is translated and the fraction of newly made polypeptides that are successfully matured into GUS tetramers. Experimental estimates of translational yield confirmed that this is indeed a source of tissue differences in GUS gene regulation. This finding also proved to be true of the B haplotype of the GUS gene. The differential regulation of special-function genes is, in general, effected transcriptionally. In contrast, the differential regulation of several "housekeeping" genes has been reported to arise from changes in mRNA maturation and/or stability. It is now apparent that translational yield, which is an aspect of protein synthesis, can also serve as a differential regulatory mechanism.

Properties of rat and mouse beta-glucuronidase mRNA and cDNA, including evidence for sequence polymorphism and genetic regulation of mRNA levels.

cDNA clones containing partial sequences for beta-glucuronidase (beta G) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize beta G mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb beta G mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the beta G gene complex. A fragment of beta G cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to beta G mRNA. This provided an extremely sensitive probe for the assay of beta G mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse beta G RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitary-dependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, beta G mRNA levels paralleled rates of beta G synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of beta G mRNA or its efficiency of translation depending on the strain of mice tested.