Calcineurin deficiency decreases inflammatory lesions in transforming growth factor beta1-deficient mice.

Transforming growth factor (TGF) beta1) is an immunoregulatory cytokine involved in self-tolerance and lymphocyte homeostasis. Tgfb1 knock-out (KO) mice develop severe multi-focal autoimmune inflammatory lesions due to [Ca(2+)]i deregulation in T cells, and die within 3 weeks after birth. Because the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1(-/-) thymocytes, and because calcineurin Abeta (CNAbeta)-deficient mice do not reject allogenic tumours, we have generated Tgfb1(-/-) Cnab(-/-) mice to address whether CNAbeta deficiency prevents T cell activation and inflammation in Tgfb1(-/-) mice. Here we show that in Tgfb1(-/-) Cnab(-/-) mice inflammation is reduced significantly relative to that in Tgfb1(-/-) mice. However, both CD4(+) and CD8(+) T cells in double knock-out (DKO) mice are activated, as revealed by up-regulation of CD11a lymphocyte function-associated antigen-1 (LFA-1), CD44 and CD69 and down-regulation of CD62L. These data suggest that deficiency of CNAbeta decreases inflammatory lesions but does not prevent activation of autoreactive T cells. Also Tgfb1(-/-) T cells can undergo activation in the absence of CNAbeta, probably by using the other isoform of calcineurin (CNAalpha) in a compensatory manner. CNAbeta-deficient T cells undergo spontaneous activation in vivo and are activated upon anti-T cell receptor stimulation in vitro. Understanding the role of calcineurin in T cell regulation should open up new therapeutic opportunities for inflammation and cancer.

Loss of the retinoblastoma tumor suppressor: differential action on transcriptional programs related to cell cycle control and immune function.

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of cis-elements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB. Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

Altered gene expression in melanocytes exposed to 4-tertiary butyl phenol (4-TBP): upregulation of the A2b adenosine receptor 1.

Exposure to phenolic agents contributes to the development of occupational vitiligo. Proposed as a causative factor for leukoderma in vivo, the para-substituted phenol 4-tertiary butyl phenol was chosen to investigate early cellular events responsible for selective disappearance of melanocytes from the epidermis of individuals sensitive to such agents. To this end, differential display of melanocyte mRNA isolated from three separate cultures was performed following a 12 h exposure of cells to 250 microM 4-tertiary butyl phenol or to vehicle alone. Fragments of cDNA representing differentially expressed messages were cloned and subsequently confirmed by reverse dot blotting. Alignment analysis revealed that the L30 ribosomal protein was upregulated by the treatment, potentially reflecting altered levels of protein synthesis in response to stress. In addition, a gene sequence upregulated following exposure to 4-tertiary butyl phenol was identified as the A2b receptor (a P1 receptor for adenosine). Differential expression of this gene was confirmed in an RNase protection assay. By reverse transcription-polymerase chain reaction, the gene was shown to be expressed in keratinocytes and fibroblasts as well. Flow cytometry confirmed differential expression in melanocytes and fibroblasts, but not in keratinocytes. Interestingly, it has been reported that P1 purinoceptor stimulation can induce apoptosis. This is in concordance with results reported elsewhere demonstrating induction of apoptosis by 4-tertiary butyl phenol in human melanocytes, as well as with morphologic changes observed in this study in cells exposed to 250 microM 4-tertiary butyl phenol for 72 h. In conclusion, differential display is useful to establish melanocyte components involved in the cellular response to phenolic agents.

Tumor necrosis factor alpha regulates CXC chemokine receptor expression and function.

The alpha chemokine family is central to the participation of neutrophils in the acute inflammatory response. These substances interact with neutrophils through two cell surface receptors, CXCR-1 and CXCR-2 (formally known as IL-8R-1 and IL-8R-2). We investigated the possible regulatory effects of tumor necrosis factor alpha (TNFalpha) pretreatment on CXCR-1 and CXCR-2. To this end, we examined these receptors with flow cytometry, radioligand binding, Northern blot analyzes, calcium mobilization, and chemotaxis experiments on human neutrophils. In flow cytometry experiments, TNFalpha pretreatment substantially decreased cell surface CXCR-2 receptor levels but showed partial recovery at 120 min. On the other hand, CXCR-1 receptor levels had a sharp decline at 15 min and maintained that level to 120 min. Northern blot analyzes showed that mRNA levels of both IL-8 receptors were essentially unchanged after 45 min of TNFalpha pretreatment, but declined markedly following 2 h of pretreatment. Chemotaxis experiments on cells treated with TNFalpha for 5-120 min showed a substantial down-regulation of chemotaxis to IL-8 and GROalpha. This was noted to be much greater than the decline in cell surface receptors. Calcium mobilization experiments revealed minimal inhibition of the IL-8-induced increase in calcium after pretreatment with TNFalpha, but the response to NAP-2 was substantially inhibited. The data demonstrate differential regulation of the IL-8 receptor.

The effect of nutritional immunomodulation on cardiac allograft survival in rats receiving mycophenolate mofetil, cyclosporine A, and donor-specific transfusion.

BACKGROUND: Immunosuppressive drugs continue to pose significant risks such as infection, toxicity, or neoplasia when used in long-term therapy. The investigation of newer and safer combined treatment strategies that decrease the need for these drugs is becoming increasingly important. Immunonutrients are known to have significant modulating effects on the immune system. Feeding with Impact, a commercially available diet enriched with arginine, omega-3 fatty acids, and RNA, recently has been shown to extend rat cardiac allograft survival when combined with a donor-specific transfusion (DST) and cyclosporine A (CsA). Because mycophenolate mofetil (MMF) is now commonly used in the clinical setting, the current study was designed to examine the effect on rat cardiac allograft survival when MMF was added to this immunosuppressive regimen. METHODS: Intra-abdominal ACI to Lewis heterotopic cardiac allografts were performed. Study groups included untreated controls and recipients receiving varying combinations of a DST (1 mL) on the day prior to engraftment, MMF 45 mg/kg/day from the day of transplant through postoperative day six, and CsA 10 mg/kg on the day prior to operation and 2.5 mg/kg from the day of transplant through postoperative day 6. Animals were fed ad libitum with Impact diet or standard lab chow. Graft survival was determined by cessation of a palpable heartbeat. RESULTS: Treatment with MMF led to a prolonged allograft survival over historical untreated controls. The combination of MMF with a donor-specific transfusion, Impact, or CsA was associated with an increase in graft survival over MMF alone. The addition of Impact to the combination of MMF and CsA resulted in further improvement. The most pronounced graft survival advantage was seen when Impact was combined with a DST and both of the immunosuppressive agents. One quarter of the animals in this group had a palpable donor heart beat at greater than 150 days, indicating functional tolerance in those animals. CONCLUSIONS: The administration of Impact diet to treatment groups in this study was associated with graft survival advantages when compared to most of the other study groups receiving a similar drug regimen and standard chow. These findings support the importance of nutritional influences on allograft survival, and highlight the potential of diet therapy when used with short courses of clinically relevant immunosuppressive drugs.

A strategy for generating consistent long-term donor-specific tolerance to solid organ allografts.

Current triple drug immunosuppression while effective, increases the risk of opportunistic infection and lymphoproliferative disorders. An alternative strategy would be the generation of donor-specific tolerance with short-term treatment. The use of donor-specific transfusions (DST) with a single brief course of cyclosporine (CsA) and rapamycin (Rapa) has produced promising results in animal models, but falls short of uniform tolerance. It was hypothesized that a DST/CsA/Rapa protocol administered in the perioperative period and redosed at one month might improve on this success in the ACI to Lewis rat heterotopic cardiac transplant model. Recipients received no treatment (group 1), a 1 ml DST intravenously (i.v.) with CsA 10 mg/kg subcutaneously (s.c.) at D-1 and CsA 2.5 mg/kg DO6D+13 (group 2), DST/CsA as dosed above with intraperitoneally (i.p.) Rapa 1 mg/kg D+36D+7 (group 3), DST/CsA/Rapa as above with all components redosed at one month (group 4), DST/CsA/Rapa with only CsA and Rapa repeated (group 5), and DST/CsA/Rapa with CsA redosed and Rapa continued indefinitely (group 6). Comparison of permanent survival (longer than 200 days) between protocols revealed groups 4-6 were significantly greater than control groups 1-3. Donor specificity was verified in group 6, where three permanent survivors received a second cardiac allograft from a Buffalo rat donor and rejected these grafts almost as quickly as untreated strain pair matched controls 21 +/- 1 days vs 30.3 +/- 5 days. Animals from group 6 displayed a greatly reduced mixed lymphocyte response to ACI cells but not to third-party cells. The percentage of T cells producing cytokines was reduced and shifted toward Th-2 type cytokines (IL-4). Thus, a repeated cycle of this brief DST/CsA/Rapa treatment appears to generate consistent permanent graft survival (up to 91%) that exceeds previously studied tolerance inducation protocols and is donor specific.

A novel flow cytometric method for quantifying phagocytosis of apoptotic cells.

Many eukaryotic cell types are capable of specific recognition and phagocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these mechanisms, we designed a novel fluorescence-based method to quantify phagocytosis in vitro using endothelial cell engulfment of apoptotic cells as a model. The B-cell line WEHI-231 was labeled with the fluorophore 5-(&-6)-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA) and then induced to undergo apoptosis by crosslinking cell surface immunoglobulin. An endothelial cell line was subsequently allowed to ingest these TAMRA-labeled apoptotic lymphocytes. After 24 h, nonbound lymphocytes were removed and the mono-layers were dissociated. Any nonphagocytosed lymphocytes that remained tightly bound to the endothelial cells were then indirectly immunofluorescein labeled for the pan leukocyte-specific marker CD45. Flow cytometric analysis of the cells distinguished three endothellal cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA+ CD45+); 2) endothelial cells containing phagocytosed apoptotic lymphocytes (TAMRA+ CD45-); and 3) endothelial cells that were not associated with lymphocytes. The identification of these populations was verified by confocal microscopy of sorted cells. The method described herein will facilitate detailed studies on phagocytic recognition of apoptotic cells and should have broad applications to other phagocytic cell systems.

Synthesis of interleukin-1 alpha and beta by normal human melanocytes.

Normal human melanocytes and melanoma cells have been reported to produce several cytokines. Previously we demonstrated that neonatal human melanocyte proliferation and tyrosinase activity are inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, and interleukin-6. We have now also shown that interleukin-1 beta induces an inhibiting effect on neonatal melanocyte tyrosinase activity with little effect on melanocyte proliferation. We investigated the ability of neonatal and adult human melanocytes to synthesize interleukin-1 alpha and beta. By immunocytochemistry, using monoclonal antibodies against interleukin-1 alpha and beta, we observed that neonatal and adult melanocytes stain positively for both cytokines. Flow-cytometric analysis revealed that the percentage of melanocytes positive for interleukin-1 alpha was always greater than that for interleukin-1 beta. The ability of neonatal and adult melanocytes to synthesize interleukin-1 alpha and beta was further confirmed using the polymerase chain reaction. These results clearly indicate that human melanocytes synthesize interleukin-1 alpha and beta, and that these cytokines may function as autocrine and/or paracrine regulators of cells in the epidermis.

Alterations of neutrophil responses to tumor necrosis factor alpha and interleukin-8 following human endotoxemia.

Interleukin-8 (IL-8), a neutrophil chemoattractant and activating cytokine, has been implicated as a proinflammatory mediator in gram-negative sepsis. In vitro data support the notion of IL-8 as an endothelial adherence inhibitor. To evaluate this issue, we infused six volunteers with reference endotoxin and measured plasma levels of IL-8, neutrophil tumor necrosis factor alpha (TNF-alpha) receptors, TNF-alpha-induced adherence to fibronectin, and neutrophil chemotaxis to IL-8 and other attractants. We found that, at 3 h postinfusion, IL-8 but not TNF-alpha plasma levels were elevated. Neutrophils had shed L-selectin (mean channel fluorescence decrease, 79 +/- 9 to 49 +/- 7; P = 0.0625) and TNF-alpha receptors (decrease in number of receptors per cell, 1,596 +/- 340 to 574 +/- 93; P = 0.004). Cells were chemotactically desensitized to IL-8. TNF-alpha-induced adherence to fibronectin was suppressed from 69% +/- 5% of the phorbol myristate acetate response to 38% +/- 7% (P = 0.0154). These findings support the notion that release of IL-8 into the vascular space may be an in vivo mechanism for suppression of neutrophil accumulation at extravascular sites. L-Selectin loss would reduce the ability of neutrophils to adhere to activated endothelial cells. The specific loss of migratory response to IL-8 would impair neutrophil delivery to areas where IL-8 was the predominant chemoattractant. Loss of TNF-alpha-induced adherence to fibronectin would blunt those responses, including production of oxidants, capacitated by adherence.

Fractionation of Pneumocystis carinii developmental stages by counterflow centrifugal elutriation and sequential filtrations.

Immunomagnetic sorting, sequential filtrations, and counterflow centrifugal elutriation were compared for their ability to obtain enriched populations of Pneumocystis carinii developmental stages from infected rat-lung homogenates. Elutriation combined with sequential filtrations resulted in highly (> 95%) enriched populations of P. carinii cysts and trophozoites with excellent viability. This approach offers advantages over previously described methods of obtaining enriched P. carinii cell populations and should have important applications to research on this organism.

Immunomodulation by an inhibitor of S-adenosyl-L-homocysteine hydrolase: inhibition of in vitro and in vivo allogeneic responses.

The response of murine T cells to MHC class II determinants on allogeneic cells induces helper T cell activation and the development of cytotoxic T cells. We have recently established that an S-adenosyl-L-homocysteine hydrolase inhibitor, (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (MDL 28,842), is a potent immunosuppressive agent which selectively inhibits T cell activation. In this report we characterize the effect of MDL 28,842 on in vitro and in vivo models of transplant rejection. In vitro, MDL 28,842 inhibited the generation of cytotoxic T cells in the murine mixed lymphocyte reaction with an IC50 of less than 0.1 microM. MDL 28,842 (1.0 microM) totally inhibited the generation of cytotoxic T cells when added up to 3 days after the initiation of culture with no apparent cell toxicity. In vivo, MDL 28,842 given by gavage at 5.0, 2.5, or 1.0 mg/kg/day inhibited the increase in popliteal lymph node weight induced by injection of allogeneic spleen cells into the footpad. MDL 28,842 was also evaluated in a model of graft rejection. Skin allografts on animals given MDL 28,842 at 5 mg/kg/day (ip) for the first 6 days following transplantation survived for 12.2 days, compared to 8.7 days for control animals. Cyclosporin A (CSA) given at 5.0 mg/kg/day did not prolong graft survival. The combination of MDL 28,842 and CSA was not any more effective than MDL 28,842 alone. Based on these findings, we suggest that MDL 28,842 is useful in the prevention of allograft rejection.

Reduced PMN beta 2 integrins after trauma: a possible role for colony-stimulating factors.

The beta 2 integrins are composed of a common 95-kD beta-subunit (CD18) and one of three possible alpha-subunits: CD11a, CD11b, or CD11c. These molecules are involved in neutrophil adhesion, diapodesis, chemotaxis and phagocytosis. In this study, the effects of traumatic injury on neutrophil expression of these alpha-subunits were investigated. Neutrophils from patients with severe trauma (n = 30) were stained with fluorescent anti-CD11a, -CD11b, or -CD11c. The percentage of positive neutrophils and the mean channel fluorescence were assayed by flow cytometry. In 10 patients and 10 normals, the effects of granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on alpha-subunit expression were evaluated. Ninety-four +/- 2% (s.e.m.) of normal neutrophils were CD11a+, 89 +/- 1% were CD11b+ and 89 +/- 8% were CD11c+. Only 65 +/- 2% of patient neutrophils were CD11a+, 45 +/- 5% were CD11b+ and 8 +/- 1% were CD11c+. Culture of normal neutrophils without colony-stimulating factors resulted in reduced expression of CD11a and CD11c, but up-regulation of CD11b. Down-regulation of CD11a and CD11c was partially reversed by colony-stimulating factors (30 U/ml). CD11b receptor density was further up-regulated by GM-CSF and G-CSF. Treatment of patient neutrophils with colony-stimulating factors in culture resulted in up-regulation of alpha-subunits as well. GM-CSF appeared to have the greater effect. These results indicate that colony-stimulating factors may have a clinical role in improving beta 2 integrin expression, and suggest a use in these infection-prone patients.

The role of protein and calorie restriction in outcome from Salmonella infection in mice.

We studied the separate effects of protein and calorie restriction in mice challenged with Salmonella typhimurium, an intracellular pathogen eliminated by cell-mediated immunity. Female A/J mice (n = 73) were placed on one of eight solid diets for 3 weeks. Animals were weighed at the beginning and the end of the feeding period. Diets were adjusted by two factors. The total amount of protein in the diet was 1%, 5%, 20%, or 40% by weight. The diets were fed to half the mice in quantities of 3 g and to the other half at 1.5 g per mouse per day. At the end of 3 weeks, mice were injected intraperitoneally with bacteria and mortality was observed for 2 weeks. Mortality was related to protein intake and was significantly higher in the 1% and 5% groups (chi 2: p = .0021). However, mortality was lower in the calorie-restricted groups (chi 2: p = .0242). Although caloric intake did not affect cell-mediated immunity, the response to 2,4-dinitrofluorobenzene was greater in the low protein groups. Lymphoproliferative responses in the mixed lymphocyte response were not affected by either caloric or protein intake. Lymphoproliferative responses to both lipopolysaccharide and phytohemagglutinin were affected by dietary protein but not by caloric intake; proliferative responses were higher in the low-protein groups. We conclude that protein restriction can increase mortality in this model. On the other hand, short-term calorie restriction can improve survival.

Reduced expression of neutrophil CD11b and CD16 after severe traumatic injury.

Overwhelming sepsis continues to be a major source of morbidity and mortality in patients who have sustained severe traumatic injury. Recently, much interest has been focused on the role of the peripheral blood neutrophil (PMN) in infections in these patients. Two surface receptors, CD11b (CR3) and CD16 (Fc gamma RIII), are thought to participate in bacterial phagocytosis and are both present on greater than 85% of normal PMNs. We have previously shown that cells that lack both of these receptors have markedly reduced phagocytic function. The purpose of this study was to determine the effect of severe trauma on the expression of these PMN receptors. Twenty severe trauma patients, age 19-70 years, presenting with an initial APACHE II score of greater than or equal to 10 were arbitrarily divided into two groups to define severity of injury: Group A, initial APACHE II of 10-18 (n = 11) and Group B, initial APACHE II of 19-25 (n = 9). Blood was obtained on admission, on Day 3, and weekly thereafter. PMNs were stained with fluorochrome-labeled monoclonal antibodies directed against CD11b and CD16 and then analyzed by flow cytometry. Controls consisted of 14 normal adults, age 20-65 years. The percentage and absolute numbers of CD11b+/CD16+ PMNs were determined for each patient or control sample. ANOVA and multiple comparison of variables (P = 0.05) were performed for each week. Values for Group A were different from controls at Weeks 0, 1, and 3. Values for Group B were significantly lower than those of controls at all weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Pneumocystis carinii cysts by flow cytometry.

Pneumocystis carinii cysts were separated as enriched populations from suspensions of lung homogenate obtained from infected rats containing all developmental stages of this organism. Isolation of cyst populations was achieved by incubating filtered lung homogenate with the fluorescent phospholipid analog 1-palmitoyl-2-C6-NBD-phosphatidylcholine. Whereas cysts did not fluorescence as a result of outer-wall restraint of lipid integration, trophozoites and young intermediate stages readily incorporated the fluorescently labeled lipid analog into their outer membrane. The two distinct labeling patterns displayed by cysts and other developmental phases of P. carinii constitute a novel, easy, and reproducible means of isolating cysts from infected lung homogenate by flow cytometry.

Distribution and survival of Escherichia coli translocating from the intestine after thermal injury.

The present investigation was performed to study the kinetics of tissue distribution and deposition of Escherichia coli and endotoxin translocating from the intestine after thermal injury. Escherichia coli was grown in the presence of 14C glucose and both labeled bacteria and endotoxin prepared from the labeled bacteria were used as translocation probes. Escherichia coli (10(8) to 10(10) bacteria) and E. coli endotoxin (100 micrograms per animal) were gavaged into the stomach immediately before a 30% burn injury was inflicted in mice. Animals were killed 1, 4 and 24 hours after burn injury. Translocation occurred extensively within 1 hour after burn injury. Expressed as amount of radioactivity per gram of tissue, translocation was greatest in the mesenteric lymph node (MLN) followed by spleen, lung, and liver. Translocation of endotoxin was similar to translocation of intact bacteria, with the exception that less radioactivity could be found in the peritoneal cavity and more in the liver. Both intact E. coli and endotoxin translocated directly through the intact bowel wall. Killing of bacteria was greatest in the MLN and spleen, approximating 95% to more than 99% of translocating bacteria. Killing efficiency was lowest in the lungs. It is concluded that estimation of translocation by viable bacterial counts in tissues grossly underestimates the extent of translocation of bacteria and ignores the extent of translocation of endotoxin. Translocation of endotoxin may have biologic significance that is independent of and in addition to translocation of intact bacteria.

Neutral surface aminopeptidase activity of human tumor cell lines.

Seven human tumor cell lines were studied for their neutral surface aminopeptidase (AP) activity. The activity was shown to exist on all cell lines to varying degrees. The neutral AP activity of the cell lines had similar Km values and were affected by the same inhibitors as those reported for AP's of peripheral blood lymphocytes (PBL, Refs. 1 and 2). However, a difference was seen in the Vmax values of the various cell lines. These values were shown to correlate (r = 0.767, P less than 0.05) with cell surface area.

The process of microbial translocation.

The process of microbial translocation was studied using Candida albicans, Escherichia coli, or endotoxin instilled into Thiry-Vella loops of thermally injured guinea pigs and rats. Translocation of C. albicans occurred by direct penetration of enterocytes by a unique process different from classical phagocytosis. Translocation between enterocytes was not observed. Internalization was associated with a disturbance of the plasma membrane and brush border, but most internalized organisms were not surrounded by a plasma membrane. Passage of the candida into the lamina propria appeared to be associated with disruption of the basal membrane with extrusion of cytoplasm of the cell and candida. Organisms in the lamina propria were commonly phagocytized by macrophages but also were found free in lymphatics and blood vessels. Translocation of E. coli and endotoxin also occurred directly through enterocytes rather than between them, but translocated endotoxin diffused through the lamina propria and muscular wall of the bowel wall by passing between rather than through the myocytes. These descriptive phenomena provide new insight into the role of the enterocyte and intestinal immune cells in the translocation process.

Flow cytometric analysis of neutrophil subsets in thermally injured patients developing infection.

The expression of CD11b (CR3, complement receptor type three), CD16 (FcR, Fc IgG receptor), and CD35 (CR1, complement receptor type one) on neutrophils obtained from thermally injured patients was examined using immunofluorescence and flow cytometry. Because defects in neutrophil function have been related to an increased risk of infection and death following thermal injury, we compared changes in neutrophil subpopulations following thermal injury with the onset of infection. Neutrophils from 34 patients with large thermal injuries were monitored weekly for CD11, CD16, and CD35. Changes in the cell surface antigens over time were compared with the incidence of bacteremia and pneumonia. Although the percentages of CD16+ CD11+ neutrophils were suppressed in almost all patients, the changes which occur in each individual patient rather than the actual values appear to be of major importance. Patients developing bacteremia or pneumonia displayed a significant reduction in both the percentage and absolute number of CD16+ CD11+ neutrophils compared to their preinfection values. The values did not increase until the infections were completely cleared. Patients remaining free of bacteremia or pneumonia usually had lower than normal percentages of CD16+ and CD11+ neutrophils with no predictable pattern being noted. The percentage of CD35+ neutrophils dropped within 1 week following thermal injury in all patients but did not correlate with the onset of infections.