Harold N. Rode

Delayed Hypersensitivity: Indicator of Acquired Failure of Host Defenses in Sepsis and Trauma

Primary failure of host defense mechanisms has been associated with increased infection and mortality. Anergy, the failure of delayed hypersensitivity response, has been shown to identify surgical patients at increased risk for sepsis and related mortality. The anergic and relatively anergic patients whose skin tests failed to improve had a mortality rate of 74.4%, whereas those who improved their responses had a mortality rate of 5.1% (P < 0.001). This study documents abnormalities of neutrophil chemotaxis, T-lymphocyte resetting in anergic patients and the effect of autologous serum. These abnormalities may account for the increased infection and mortality rates in anergic patients. Skin testing with five standard antigens has identified 110 anergic (A) or relatively anergic (RA) patients in whom neutrophil chemotaxis (CTX) and bactericidal function (NBF), T-lymphocyte rosettes, mixed lymphocyte culture (MLC), cell-mediated lympholysis (CML), and blastogenic factor (BF) were studied. The MLC, CML and BF were normal in the patients studied, and were not clinically helpful. Neutrophil CTX in 19 controls was 117.5 ± 1.6 u whereas in 40 A patients, neutrophils migrated 81.7 ± 2.3 u and in 15 RA patients 97.2 ± 3.8 u (P < 0.01). In 14 patients whose skin tests converted to normal, neutrophil migration improved from 78.2 ± 5.4 u to 107.2 ± 4.0 u (P < 0.01). Incubation of A or control neutrophils in A serum reduced migration in A patients from 93 ± 3.7 u to 86.2 ± 3.5 u (P < 0.01) and in normals from 121.2 ± 1.6 u to 103.6 ± 2.6 u (P < 0.001). The per cent rosette forming cells in 66 A patients was 42.5 ± 3.1 compared to 53.6 ± 2.8 in normal responders (P < 0.02). Incubation of normal lymphocytes in anergic serum further reduced rosetting by 30%. Restoration of delayed hypersensitivity responses and concurrent improvement in cellular and serum components of host defense were correlated with maintenance of adequate nutrition and aggressive surgical drainage.

Estimating mortality risk in preoperative patients using immunologic, nutritional, and acute-phase response variables.

We measured the delayed type hypersensitivity (DTH) skin test response, along with additional variables of host immunocompetence in 245 preoperative patients to determine which variables are associated with septic-related deaths following operation. Of the 14 deaths (5.7%), 12 were related to sepsis and in 2 sepsis was contributory. The DTH response (p less than 0.00001), age (p less than 0.0002), serum albumin (p less than 0.003), hemoglobin (p less than 0.02), and total hemolytic complement (p less than 0.03), were significantly different between those who died and those who lived. By logistic regression analysis, only the DTH skin test response (log likelihood = 41.7, improvement X2 = 6.24, p less than 0.012) and the serum albumin (log likelihood = 44.8, improvement X2 = 17.7, p less than 0.001) were significantly and independently associated with the deaths. The resultant probability of mortality calculation equation was tested in a separate validation group of 519 patients (mortality = 5%) and yielded a good predictive capability as assessed by (1) X2 = 0.08 between observed and expected deaths, NS; (2) Goodman-Kruskall G statistic = 0.673) Receiver-Operating-Characteristic (ROC) curve analysis with an area under the ROC curve, Az = 0.79 +/- 0.05. We conclude that a reduced immune response (DTH skin test anergy) plus a nutritional deficit and/or acute-phase response change are both associated with increased septic-related deaths in elective surgical patients.

Macrophages in the mixed leukocyte culture reaction (MLC)

Abstract Two aspects of the mixed leukocyte culture reaction have been investigated. In the first study the capacity of two highly purified adherent cell populations, one consisting of neutrophils the other of macrophages, to support the reactivtiy of purified lymphocytes was compared. Activity was entirely confined to the latter preparation. In the second study, the ability of platelets, neutrophils, lymphocytes and macrophages to provoke a MLC reaction was studied. Macrophages were found to be 10 times as efficient in eliciting a reaction as an equal number of lymphocytes. Neutrophils and platelets appeared to be devoid of stimulating capacity. This lack of stimulation was shown to be due to a true deficiency in stimulating capacity rather than to an inhibition obtained by high concentrations of neutrophils and platelets. The two functions of the macrophage in MLC, its auxiliary helper role, and the elicitation of the reaction, may be dependent on one and the same mechanism.

In vivo and in vitro humoral immunity in surgical patients

In vivo and in vitro humoral immunity was studied in surgical patients. Laboratory controls (LC), delayed type hypersensitivity (DTH) skin test reactive (HR), and anergic (HA) patients were immunized with tetanus toxoid. Maximum in vivo antibody levels occurred 14 days after immunization. Eighty-six, 47, and 17% of LC, HR, and HA subjects, respectively, showed a positive response (xl22 = 21.1 with Yates, p < 0.0005). Peak in vitro antibody production in unstimulated lymphocyte cultures occurred at day 6 after immunization. Antibody responses in vitro were reduced in all surgical patients, worst in HA, and correlated quantitatively with in vivo antibody responses at day 14 (Spearman rank correlation = 0.794, p < 0.001). Total IgG production in vitro was not decreased; 595, 1080, and 1538 ng IgG/culture were produced by LC, HR, and HA, respectively. These data demonstrate decreased in vivo and in vitro humoral immunity in all surgical patients, worst in those with decreased DTH responses. There is a kinetic and quantitative correlation between in vivo and in vitro responses, the latter being a biologic reflection of the integrity and magnitude of the in vivo process. Finally, failure to produce specific antibody is not due to failure of total IgG synthesis.

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.

The walk-in anergic patient. How best to assess the risk of sepsis following elective surgery.

This prospective study evaluated host resistance in a surgical population who walked into the hospital for elective surgery. Patients were stratified into Hospital Reactive (HR, n = 19) if they reacted to two or more of five recall skin test antigens and Walk-in Anergic (WA, n = 26) if they did not react to the antigens. The WA patients were slightly older (74.4 ±1.8 years, ± SEM versus 66.7 ± 2.7 p < 0.05). Diagnosis in the HR and WA group were: tumors 13/19 versus 21/26, diverticulitis 3/19 versus 0/19, and miscellaneous 3/19 versus 5/26. Twenty-five laboratory normal controls (LN) were also studied. There were no significant differences in the following parameters between the HR and WA groups: stage of disease; hemoglobin; circulating leukocyte count; polymorphonuclear cell counts; total lymphocyte counts (both groups lower than LN, p < 0.05), monocyte counts (both higher than LN, p < 0.05); per cent E-rosettes and lymphocyte blastogenesis to mitogens (phytohem-alutin, concanavalin-A) and antigens (purified protein derivative and tetanus); phagocytosis of preopsonised Staphylococcus aureus 502A, at 5, 10, and 20 minutes; alpha, beta, and gamma globulins; C3, and total hemolytic complement (CH50) levels; C-reactive protein; and ANA and DNA levels. The HR group demonstrated an increase in the rate of killing of Staphylococcus 502A at 10, 20, 40, and 80 minutes compared to the LN group but the WA group did not show this augmentation (p < 0.001). The serum albumins were: LN = 4.46, HR = 3.98, WA = 3.43 g/dl (p < 0.05). Degree and duration of surgery was the same in the HR and WA groups. There were no major sepsis episodes (bacteremia or proven intracavitary abscess) in the HR patients versus 25% in the WA patients (p < 0.05). There was one death (6%, pulmonary embolus) in the HR group and 8 (40%) in the WA group (p < 0.05). Antibiotic prophylaxis was equal but the WA patients received therapeutic antibiotics more frequently (65% versus 11% p < 0.05). Of all the host immunocompetence tests measured in this study, the delayed type hypersensitivity skin test response and the serum albumin were variables abnormal between the survivors and those who died.

Skin window chambers, a novel method for recovering the cells involved in delayed-type hypersensitivity

A method is described for the recovery of cells involved in the delayed-type hypersensitivity (DTH) reaction in man. A small plastic chamber was placed over DTH sites from which the stratum corneum had been removed by tape stripping. The chambers were then filled with serum. Analysis of cells harvested at 24 and 48 h has shown an antigen-dependent increment in mononuclear and polymorphonuclear cells in donors with a DTH response. T cell clones were established from the recovered cells; these will provide a useful adjunct to a functional analysis of cells participating in the DTH reaction.

FUNCTIONAL ORGANIZATION OF THE LYMPHOCYTE PLASMA MEMBRANE RELATING TO CELL ACTIVATION

Publisher Summary This chapter discusses the initiation of lymphocyte activation. The chapter presents some studies in which the aim was to observe composition of the microenvironment of a receptor and its putative changes upon the binding of a mitogen. The model used in these studies consisted of calf thymus lymphocytes as a source of pure T lymphocytes and concanavalin A (Con A) as the mitogen. This model offers several advantages and disadvantages. It is easy to get great numbers of fairly homogeneous cells, a great proportion of which can be activated with the lectin Con A. On the other hand, the definite nature of the activating receptor is largely unknown. Lectins such as Con A bind to a variety of glycoproteins or glycolipids; however, the activation appears to be mediated only by a small proportion of binding sites. The activating receptors have been shown to be glycoproteins, which are distinct from the bulk binding sites by having a higher affinity for the mitogen. Thus, the different affinity of glycoproteins is the only criterion distinguishing mitogen receptors from bulk binding sites.

Lymphocyte activation by purified HLA-DR molecules fused into autochthonous “stimulating cells”

Affinity-purified Ia molecules derived from the Daudi cell line were reconstituted into vesicles with Sendai virus envelope glycoproteins. These vesicles inserted into human peripheral leukocytes could induce stimulation of autologous lymphocytes, as measured by thymidine uptake, 6 days later. It is suggested that this method could provide a means to study allostimulation at the molecular level.

FUNCTIONAL MOSAICISM OF THE LYMPHOCYTE PLASMA MEMBRANE

Publisher Summary This chapter explores the functional mosaicism of the lymphocyte plasma membrane. Lymphocytes are activated to grow and divide when only a distinct proportion of plasma membrane-binding sites interacts with mitogens such as the lectin concanavalin A (ConA). To test whether binding sites responsible for cell activation are associated with specialized areas of the plasma membrane, calf thymocytes were disrupted by the nitrogen cavitation method and the microsomal membranes were isolated in a study described in the chapter. These vesicles, which are more than 90% derived from the plasma membrane, were subjected to affinity chromatography on ConA-sepharose. The results demonstrate that the plasma membrane of lymphocytes is not completely homogeneous. The areas of the plasma membrane that carry mitogen receptors, that is, high affinity binding sites, are distinct from the bulk membrane with regard to their enzymatic activities, suggesting a functional mosaicism in the vicinity of mitogen receptors.