Lymphocyte Activation

Abstracts of papers presented at the Nordic Symposium, 1st and 2nd of December, 1972, in Helsinki, Finland.
The meeting was sponsored by:
Minerva Institute for Medical Research, Folkhälsen Institute for Genetics, Swedish Cancer Society, Study Group for Mitogenic Substances, and arranged by: Theodor H. Weber & Valdemar T. Skoog.
The topics covered were: general and clinical aspects of lymphocyte activation and separation and surface characteristics of T and B lymphocytes.     

Activation of lymphocytes

The technique involved in studying the activation of lymphocytes in the resting form, and their recognition as dividing and functional cells was studied, using phase contrast and agar as well as fluid culture. Standardization of technical methods was found to be essential, and the effect of variables was studied. Lymphocytes from human umbilical cord vein blood were found to be spontaneously activated. Infestation by microfilaria either diminished or entirely inhibited activation. The significance of lymphocytic cohesion was considered and the formation of colonies of activated lymphocytes on agar is described. The effects of non-human vertebrate lymphocytes and of human cells other than lymphocytes were studied. Spontaneous activation of abnormal lymphocytes was noted.     

B Cell Activation

We consider that 2 'signals’are essential for B cell activation. 'Signal 1’is a consequence of appropriate latticing of surface immunoglobulin receptors. 'Signal 2’can be mediated by a variety of agents. Signal 1 alone (i.e. surface Ig aggregation) leads to a state of B cell tolerance. Appropriate levels of signal 1 and signal 2 lead to immune induction. Inappropriate combinations of either signal such that one is in‘excess’can direct B cells towards a state of tolerance. T cells are felt to fulfil two roles, (a) They release antigen specific molecules capable of creating a lattice of antigenic determinants on a B cell surface, (b) They are responsible in interacting with macrophages for the production of NSF or Signal 2. (c) The B cell Ig-antigen-T cell receptor complex can serve to localise the effects of NSF to the region of the antigen specific B cell, and thus provide a safeguard against autoallergy. Thymus independent antigens are capable of providing both types of signal.     

Echinacea-Induced Macrophage Activation

Public interest in Echinacea is growing rapidly. Unfortunately, there is little scientific evidence to support claims of efficacy of this widely used botanical, and little information about potential mechanism of action. This study examines the ability of Echinacea to upregulate macrophage function and begins to elucidate the mechanism of Echinacea-induced macrophage activation. Murine peritoneal macrophages were cultured with E. purpurea extracts enriched for plant polysaccharide (EP). ELISA was used to measure cytokine production. MAPKs were blocked using specific inhibitors, and Western blotting used to identify phosphorylated proteins involved in signal transduction. To examine in vivo efficacy, EP was administered orally and Listeria monocytogenes given i.v. Mice were sacrificed three days post-infection to determine bacterial load in the spleen. We demonstrate that an endotoxin-free EP extract activates the innate immune response, stimulating production of IL-6, TNF, IL-12, and NO from macrophages in vitro. Along with evidence of enhanced macrophage function, we found that oral EP reduces bacterial burden during infection by Listeria monocytogenes, demonstrating its efficacy in vivo. EP initiates a signaling cascade within macrophages through both TLR4-dependent and -independent mechanisms, involving ERK, p38 and JNK, and ultimately the activation of NF-κB.     

Activation of gamma-interferonogenesis

Production of gamma-interferon was increased considerably by priming with homologous gamma- or alpha-interferons as well as by T-activin or vitamins of group B. The factors under study were effective both in the population of mononuclears and in the total fraction of nucleated cells of the donor blood. The maximal yields of gamma-interferon, up to 10,000 IU/ml, could be obtained by priming with gamma-interferon in a dose of 5 IU/ml or alpha-interferon in a dose of 1000 IU/ml. T-activin and vitamin B12 also enhanced production 8-10-fold. The combined use of priming, T-activin, and vitamin B12 in various combinations gave no further increase in the yield of activity.     

Murine APC Activation in the Xenogeneic MLC

Purified human T cells proliferate in response to direct and indirect presentation of human alloantigens. However, until recently it was believed that human T cells could respond only indirectly to murine xenoantigens. We recently used the mixed leucocyte culture (M LC) to demonstrate that purified human T cells proliferate in response to direct presentation of murine xenoantigens by murine antigen-presenting cells (APC) in the presence of human cytokines. We suggested that cytokines might function poorly across species barriers. In this study, we demonstrate that although proliferation occurs in the presence of exogenously added cytokines, the precursor frequency of responding human T cells is much lower in a xenogeneic than in an allogeneic MLC. We demonstrate that human T cells also proliferate in response to murine APC in the presence of murine cytokines, and murine cytokines augment the proliferative response seen in a human anti-human MLC, ruling out the possibility that an absolute cytokinc incompatibility exists between these species. We show that exogenously added human IL-1 causes maximal proliferation of human T cells in response to murine xenoantigens only when added early in the culture. We further demonstrate that murine APC preincubatcd in human rIL-1, and washed extensively, prior to use as stimulating cells, cause human T-cell proliferation without the need for exogenously added cytokines. Finally, we noted that during interactions of human T cells and murine APC, little to no IL-1 is produced, whereas after the addition of exogenous IL-1, a marked increase in the production of IL-1 is seen. These data suggest that during interactions between human T cells and murine APC, the murine cells do not receive adequate stimulation to produce sufficient costimulatory signals to allow proliferation of the human T cells.     

Beef-Induced Anaphylaxis Confirmed by the Basophil Activation Test

Beef allergies are relatively rare, especially in adults. However, clinical manifestations can vary from urticaria, angioedema, anaphylaxis to gastrointestinal symptoms. Currently available tests, such as skin testing or in vitro determination of beef-specific immunoglobulin E (IgE), do not provide an accurate diagnosis of beef allergy. The recent development of the basophil activation test (BAT) presents a new opportunity for the diagnosis of food allergies. Here, we report a 37-year-old woman with a history of recurrent generalised urticaria, nausea, vomiting and hypotension after ingestion of beef, suggesting a beef allergy. Although the skin prick test and serum specific IgE to beef, pork and milk allergens showed negative results using commercial kits, the BAT showed significant upregulation of CD203c in a dose-dependent manner compared to both non-atopic and atopic controls. To our knowledge, this is the first case study of beef allergy consisting of a non-IgE-mediated reaction. The detection of food allergies using direct basophil activation is suggested to complement conventional diagnostic tests.     

Lymphocyte Activation by the Fc Region of Immunoglobulins

The Fc region of Ig is required for numerous biological effector functions which include: 1) opsonization, 2) anaphylaxis, 3) C fixation, 4) catabolism of the Ig molecule, 5) FcR binding, and 6) immune regulation. To this latter point, the cellular and subcellular events involved in immune regulation by IC and Fc fragments of Ig have been the focus of numerous investigations.

Characterization of cyanogen bromide cleavage fragments from a human IgG₁ myeloma protein indicates that one biologically-active site is found in residues 335–357 of the CH₃ domain of the molecule. Synthesis of the biologically-active region resulted in a peptide, termed p23, which stimulates mouse and human B cells to secrete polyclonal Ig and activates AA metabolic pathways. In contrast to these findings, p23 is unable to induce B cell proliferation or IL-1 secretion from macrophages. Analysis of data obtained with overlapping peptides, based on p23, suggests that the minimal active sequence needed for B cell differentiation is leu-pro-pro-ser-arg (residues 351–355). In contrast, only p23 or p23 minus the carboxyterminal glu₃₅₆ and glu₃₅₇ were able to induce PGE release.

Release of biologically-active peptides derived from the Fc region of Ig into the cellular microenvironment may form the nucleus of a nonspecific in vivo Immunoregulatory network. The specificity of peptide regulatory activities could reside in their effectiveness at high concentrations in the cellular microenvironment. The interaction of Fc region peptides with receptors on B cells, T cells, and macrophages/monocytes could result in a dynamic control of immune reactivity.     

Activation of the visual cortex in motivated attention

Functional activation (measured with fMRI) in occipital cortex was more extensive when participants view pictures strongly related to primary motive states (i.e., victims of violent death, viewer-directed threat, and erotica). This functional activity was greater than that observed for less intense emotional (i.e., happy families or angry faces) or neutral images (i.e., household objects, neutral faces). Both the extent and strength of functional activity were related to the judged affective arousal of the different picture contents, and the same pattern of functional activation was present whether pictures were presented in color or in grayscale. It is suggested that more extensive visual system activation reflects "motivated attention," in which appetitive or defensive motivational engagement directs attention and facilitates perceptual processing of survival-relevant stimuli.     

Lymphocyte Activation by the Fc Region of Immunoglobulins

The Fc region of Ig is required for numerous biological effector functions which include: 1) opsonization, 2) anaphylaxis, 3) C fixation, 4) catabolism of the Ig molecule, 5) FcR binding, and 6) immune regulation. To this latter point, the cellular and subcellular events involved in immune regulation by IC and Fc fragments of Ig have been the focus of numerous investigations.

Characterization of cyanogen bromide cleavage fragments from a human IgG₁ myeloma protein indicates that one biologically-active site is found in residues 335-357 of the CH₃ domain of the molecule. Synthesis of the biologically-active region resulted in a peptide, termed p23, which stimulates mouse and human B cells to secrete polyclonal Ig and activates AA metabolic pathways. In contrast to these findings, p23 is unable to induce B cell proliferation or IL-1 secretion from macrophages. Analysis of data obtained with overlapping peptides, based on p23, suggests that the minimal active sequence needed for B cell differentiation is leu-pro-pro-ser-arg (residues 351-355). In contrast, only p23 or p23 minus the carboxyterminal glu₃₅₆ and glu₃₅₇ were able to induce PGE release.

Release of biologically-active peptides derived from the Fc region of Ig into the cellular microenvironment may form the nucleus of a nonspecific in vivo Immunoregulatory network. The specificity of peptide regulatory activities could reside in their effectiveness at high concentrations in the cellular microenvironment. The interaction of Fc region peptides with receptors on B cells, T cells, and macrophages/monocytes could result in a dynamic control of immune reactivity.