Ellis Lightfoot

CD8αβ T Cells Are Not Essential to the Pathogenesis of Arthritis or Colitis in HLA-B27 Transgenic Rats

The class I MHC allele HLA-B27 is highly associated with the human spondyloarthropathies, but the basis for this association remains poorly understood. Transgenic rats with high expression of HLA-B27 develop a multisystem inflammatory disease that includes arthritis and colitis. To investigate whether CD8αβ T cells are needed in this disease, we depleted these cells in B27 transgenic rats before the onset of disease by adult thymectomy plus short-term anti-CD8α mAb treatment. This treatment induced profound, sustained depletion of CD8αβ T cells, but failed to suppress either colitis or arthritis. To address the role of CD8α+β− cells, we studied four additional groups of B27 transgenic rats treated with: 1) continuous anti-CD8α mAb, 2) continuous isotype-matched control mAb, 3) the thymectomy/pulse anti-CD8α regimen, or 4) no treatment. Arthritis occurred in ∼40% of each group, but was most significantly reduced in severity in the anti-CD8α-treated group. In addition to CD8αβ T cells, two sizeable CD8α+β− non-T cell populations were also reduced by the anti-CD8α treatment: 1) NK cells, and 2) a CD4+CD8+CD11b/c+CD161a+CD172a+ monocyte population that became expanded in diseased B27 transgenic rats. These data indicate that HLA-B27-retricted CD8+ T cells are unlikely to serve as effector cells in the transgenic rat model of HLA-B27-associated disease, in opposition to a commonly invoked hypothesis concerning the role of B27 in the spondyloarthropathies. The data also suggest that one or more populations of CD8α+β− non-T cells may play a role in the arthritis that occurs in these rats.

Major Burn Trauma In Rats Promotes Cardiac And Gastrointestinal Apoptosis

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43+/-1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringers solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and +/-dP/dt(max). Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

Effect of a bradykinin antagonist on the local inflammatory response following thermal injury

Partial and full thickness burns with intervening zones of stasis were created on the backs on New Zealand White rabbits (n = 23). Either saline or the bradykinin receptor antagonist, NPC 17731, was administered. Skin blood flow was measured hourly using a laser Doppler blood flowmeter. After 4 h skin samples were harvested for assessment of tissue oedema (wet/dry weights) and leucocyte accumulation (immunohistochemistry). Statistical analysis was performed using Analysis of variance (ANOVA) and Mann-Whitney U test with a level of significance at P < 0.05. It was found that blood flow was decreased postburn in all groups. Bradykinin antagonist resulted in increased blood flow in partial thickness burns and zones of stasis compared to saline-treated animals (P < 0.05). Pretreatment with bradykinin antagonist showed reduced tissue oedema in full thickness burns (P < 0.05). No significant difference was observed in leucocyte accumulation between both groups. These data suggest a role for bradykinin in the pathogenesis of postburn microvascular changes which is independent of leucocyte-mediated injury.

Inflammation, immune reactivity, and angiogenesis in a severe combined immunodeficiency model of rheumatoid arthritis

Severe combined immunodeficiency (SCID) mice were engrafted with rheumatoid arthritis (RA) synovium and evaluated to determine whether RA synovial morphology and function were maintained in the RA-SCID grafts. The four major components of RA synovitis, inflammation, immune reactivity, angiogenesis, and synovial hyperplasia persisted in RA-SCID grafts for 12 weeks. Retention of chronic inflammatory infiltrates was demonstrated by histological evaluation and by immunohistology for CD3, CD20, and CD68. Staining for CD68 also revealed that the grafts had undergone reorganization of the tissue, possibly as a result of fibroblast hyperplasia. Immune and inflammatory components were confirmed by the detection of human immunoglobulins and human interleukin-6 in serum samples obtained from grafted animals. Human blood vessels were detected by dense expression of CD31. Small vessels persistently expressed the vitronectin receptor, alpha v beta 3, a marker of angiogenesis. All vessels expressed VAP-1, a marker of activated endothelial cells. Finally, the grafts retained the ability to support immigration by human leukocytes, as demonstrated by the functional capacity to recruit adoptively transferred 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester-labeled T cells. T cells entering the RA-SCID grafts became activated and produced interferon-gamma, as detected by reverse transcriptase-polymerase chain reaction analysis. These studies demonstrate that the RA-SCID model maintains many of the phenotypic and functional features of the inflamed RA synovium.

Alterations in leukocyte adhesion molecule expression after burn injury

OBJECTIVE To determine if thermal injury alters the expression of leukocyte adhesion molecules. DESIGN This is a controlled experimental animal study. MATERIALS AND METHODS Partial thickness burns were created on the backs of New Zealand White rabbits. At 30 minutes, 2 hours, 4 hours, and 24 hours after burn, skin was harvested for immunohistochemistry. Monoclonal antibodies were used to study changes in intercellular adhesion molecule 1 (ICAM-1), E-selectin, and leukocyte CD11a. Staining was graded on a scale of 0 to 4. MEASUREMENTS AND MAIN RESULTS ICAM-1 was significantly decreased at 24 hours after burn (p < 0.007, Wilcoxon signed rank test). CD11a was increased at 30 minutes (p < 0.02), 2 hours (p < 0.02), and 24 hours (p < 0.006). E-selectin was increased at 2 hours (p < 0.03). CONCLUSION Thermal injury alters the expression of leukocyte adhesion molecules.

Effect of inhibiting leukocyte integrin (CD18) and selectin (L-selectin) on susceptibility to infection with Pseudomonas aeruginosa.

Leukocyte (WBC) adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury. The process of leukocyte adherence is mediated by both the integrin and selectin families of molecules, and their interaction with specific endothelial ligands. Antibodies directed against the leukocyte integrin CD18 and L-selectin have been developed and functionally inhibit leukocyte adherence in models of inflammatory injury. We asked the question: Does inhibition of leukocyte adherence by administration of monoclonal antibody directed against either CD18, integrins (R15.7, R7.1) or against L-selectin (DREG 200) increase susceptibility to infection? New Zealand white rabbits were shaved and injected subcutaneously on their dorsum with Pseudomonas aeruginosa (ATCC#27853) at two sites each of 10(8) and 10(7) colony forming units. Animals were monitored with daily determination of weight, temperature, WBC counts, hematocrit, and killed at 1 week for determination of abscess formation. There were four blinded experimental groups: (1) Saline (2 mL/kg); (2) DREG 200 (2 mg/kg); (3) R7.1 (2 mg/kg); or (4) R15.7 (2 mg/kg). At the 10(7) and 10(8) injection sites the R15.7 group had an increased rate and size of abscess formation compared with controls. The R7.1 group had an increased rate at the 10(8) injection site. There was no significant difference in the percentage of the abscess formation or mean area between the controls and DREG 200-treated groups. We conclude that giving antibody to CD18 increased susceptibility to infection while giving antibody to L-selectin does not.

Role of CD14 in hemorrhagic shock-induced alterations of the monocyte tumor necrosis factor response to endotoxin

OBJECTIVE To determine if the shock-induced alterations in whole blood monocyte tumor necrosis factor (TNF) response are mediated by the CD14 receptor. DESIGN Prospective controlled animals experiments. MATERIALS AND METHODS New Zealand White rabbits (n = 15) were subjected to hemorrhage and resuscitation. Blood samples obtained before shock and 24, 72, and 120 hours after shock were stimulated with lipopolysaccharide in the presence or absence of the anti-CD14 monoclonal antibody, 63D3. Tumor necrosis factor was assayed using L929 cells. MEASUREMENTS AND MAIN RESULTS There are no detectable TNF activity in unstimulated blood. The CD14 inhibition resulted in a 55% reduction in baseline TNF activity. After shock, there was a marked increase in TNF activity with lipopolysaccharide stimulation. Addition of 63D3 resulted in a dose-dependent 95% reduction in TNF activity at 24 and 72 hours after shock, (p < 0.05). CONCLUSION The enhanced whole blood monocyte TNF response after hemorrhage is CD14 dependent.

The Role of CD11a/CD18–CD54 Interactions in the Evolution of Animal Models of Tissue Injury

Entry of circulating inflammatory cells into tissue plays a central role in the evolution of many forms of tissue injury (1). In many of these conditions, inflammation is a prominent feature, whereas in others tissue injury is the primary manifestation, with inflammation being less apparent. An example of the former is inflammatory arthritis, in which the signs and symptoms of inflammation predominate, although persistent inflammation results in considerable damage to articular structures. On the other hand, in burn injury, tissue injury is readily apparent, whereas an inflammatory component is less prominent. However, in both conditions, it is likely that entry of circulating inflammatory cells into the affected tissue plays a primary role in the progression of tissue injury (2–5).