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Lung CD8(+) T cells might contribute to progression of chronic obstructive pulmonary disease (COPD) indirectly via IFN-gamma production or directly via cytolysis, but evidence for either mechanism is largely circumstantial. To gain insights into these potential mechanisms, we analyzed clinically indicated lung resections from three human cohorts, correlating findings with spirometrically defined disease severity. Expression by lung CD8(+) T cells of IL-18R and CD69 correlated with severity, as did mRNA transcripts for perforin and granzyme B, but not Fas ligand. These correlations persisted after correction for age, smoking history, presence of lung cancer, recent respiratory infection, or inhaled corticosteroid use. Analysis of transcripts for killer cell lectin-like receptor G1, IL-7R, and CD57 implied that lung CD8(+) T cells in COPD do not belong to the terminally differentiated effector populations associated with chronic infections or extreme age. In vitro stimulation of lung CD8(+) T cells with IL-18 plus IL-12 markedly increased production of IFN-gamma and TNF-alpha, whereas IL-15 stimulation induced increased intracellular perforin expression. Both IL-15 and IL-18 protein expression could be measured in whole lung tissue homogenates, but neither correlated in concentration with spirometric severity. Although lung CD8(+) T cell expression of mRNA for both T-box transcription factor expressed in T cells and GATA-binding protein 3 (but not retinoic acid receptor-related orphan receptor gamma or alpha) increased with spirometric severity, stimulation of lung CD8(+) T cells via CD3epsilon-induced secretion of IFN-gamma, TNF-alpha, and GM-CSF, but not IL-5, IL-13, and IL-17A. These findings suggest that the production of proinflammatory cytokines and cytotoxic molecules by lung-resident CD8(+) T cells contributes to COPD pathogenesis.

Eotaxin participation was analyzed during types 1 and 2 lung granuloma formation induced by embolizing Sepharose beads coupled to purified protein derivative (PPD) of Mycobacterium bovis or soluble Ags derived from Schistosoma mansoni eggs. Eotaxin was monitored by protein ELISA and semiquantitative reverse-transcriptase PCR mRNA analysis. Both types 1 and 2 granulomas released eotaxin, but levels were sixfold greater (on day 4) in the type 2 than for the type 1 or foreign body granulomas. Transcripts for eotaxin, IL-4, and CCR3 (eotaxin receptor) were also enhanced during type 2 granuloma formation. Anti-IL-4 treatment impaired eotaxin mRNA in lungs with type 2 granulomas, indicating that IL-4 promoted local eotaxin expression. In vivo, anti-eotaxin treatment caused modest reductions in the size of both types 1 and 2 lesions, with negligible effect on eosinophil recruitment. Surprisingly, anti-eotaxin treatment abrogated IFN-gamma-producing cells in regional lymph nodes during the type 1 PPD response. Lymph nodes draining both types 1 and 2 lesions showed enhanced CCR3 mRNA, but this followed the time of maximum eotaxin protein and mRNA expression. Correlative, in vitro studies revealed that graded doses of eotaxin increased IFN-gamma production from PPD-sensitive regional lymph node cultures, while monocyte-chemotactic protein-1, an important macrophage chemoattractant, had the opposite effect. These findings indicate that eotaxin expression is not limited to type 2 hypersensitivity granulomas, but also promotes IFN-gamma production during mycobacterial responses.

Background: Disseminated intravascular coagulation is an established complication of acute hemolytic transfusion reactions, particularly those involving the ABO red cell (RBC) antigen system. In addition, peripheral blood white cells, particularly monocytes, have demonstrated expression of procoagulant activity (PCA) in response to inflammatory stimuli. To better define the activation of coagulation in immune hemolysis, in vitro experiments were conducted to investigate the expression of PCA by peripheral blood white cells in ABO RBC incompatibility., Study Design and Methods: Fresh group O heparinized whole blood was incubated with washed, packed group A or O RBCs. White cells were separated, washed, and lysed before assay of PCA, which was measured by a one-stage recalcified clotting time assay. Units of activity were calculated on the basis of a rabbit brain thromboplastin standard curve. Mechanisms of coagulation activation were investigated by using specific coagulation factor-deficient plasmas, blocking antibodies to tissue factor, and anti-CD11b., Results: Significant levels of white cell-associated PCA were found at 2 to 6 hours in response to incompatible (group A) RBCs, but not in response to compatible (group O) RBCs. PCA was not correlated with numbers of platelets in whole blood. Nonimmune lysis of compatible RBCs did not induce PCA. When whole blood reconstituted from washed cells and heat-inactivated plasma was incubated with incompatible RBCs, PCA and hemolysis were abrogated, which suggests that complement is a required intermediate. Protein synthesis inhibition by the addition of cycloheximide (5 mg/mL) to whole blood incubated with RBCs prevented the expression of PCA. Substitution of factor VIII-deficient plasma for normal plasma in the recalcified clotting time assay had no effect, whereas PCA was reduced by 68 percent with factor VII-deficient plasma and was unmeasurable with factor X-deficient plasma. PCA was restored by a 1-to-1 mix of normal and factor VII-deficient plasma. Incubation of samples in the PCA assay with tissue factor antibodies resulted in up to 86-percent inhibition of measured PCA. Titration of the response to the amount of tissue factor antibodies added demonstrated that maximal inhibition occurred with 0.45 mg per mL, above which no further inhibition took place. However, the addition of anti-CD11b (0.75 mg/mL) concomitantly with anti-tissue factor abolished measurable activity. This effect was independent of the amount of added protein, and anti-CD11b alone had no effect on measured activity. The addition to whole blood concomitantly with RBCs of polyclonal antibodies to tumor necrosis factor, sufficient to neutralize 2000 pg per mL, did not alter PCA expression., Conclusion: These results indicate that white cell-associated PCA is generated in whole blood in response to ABO RBC incompatibility and may contribute to disseminated intravascular coagulation in acute hemolytic transfusion reactions. Two possible cellular mechanisms are suggested, which involve tissue factor expression and the activation of factor X by a CD11b-dependent mechanism.