Hans Wigzell

The Expression of the Gene Coding for the Antibacterial Peptide LL-37 Is Induced in Human Keratinocytes during Inflammatory Disorders

The epithelia constitute a major barrier to the environment and provide the first line of defense against invading microbes. Antimicrobial peptides are emerging as participants in the defense system of epithelial barriers in general. Originally we isolated the human antimicrobial peptide LL-37 from granulocytes. The gene (CAMP or cathelicidinantimicrobial peptide) coding for this peptide belongs to the cathelicidin family, whose members contain a conserved pro-part of the cathelin type. The human genome seems to have only one gene of this family, whereas some mammalian species have several cathelicidin genes. In the present work we demonstrate up-regulation of this human cathelicidin gene in inflammatory skin disorders, whereas in normal skin no induction was found. By in situ hybridization and immunohistochemistry the transcript and the peptide were located in keratinocytes throughout the epidermis of the inflammatory regions. In addition, the peptide was detected in partially pure fractions derived from psoriatic scales by immunoblotting. These fractions also exhibited antibacterial activity. We propose a protective role for LL-37, when the integrity of the skin barrier is damaged, participating in the first line of defense, and preventing local infection and systemic invasion of microbes.

LYMPHOPENIA AND CHANGE IN DISTRIBUTION OF HUMAN B AND T LYMPHOCYTES IN PERIPHERAL BLOOD INDUCED BY IRRADIATION FOR MAMMARY CARCINOMA.

Abstract Irradiation for mammary carcinoma frequently leads to a lymphopenia lasting for at least a year. An analysis of the relative proportion of bone-marrow (B) and thymus (T) dependent lymphocytes in peripheral blood has been done in these patients. One surface marker characteristic for B lymphocytes (receptors for activated complement, EAC) and spontaneous rosette-forming capacity for sheep red blood-cells (E), a property of human T lymphocytes, have been investigated in peripheral-blood lymphocytes up to a year after radiotherapy. The ability of lymphocytes to respond to the stimulus of phytohaemagglutinin (P.H.A.) was also tested before and after irradiation was given in vivo. Thirty-four patients have been followed up by blood differentials over a year and the frequency of B and T cells analysed. A statistically significant lymphopenia was found 1 year after unilateral parastemal irradiation. Healthy controls had a mean of 32% EAC-binding lymphocytes (B cells) and 60% E-binding lymphocytes (T cells). After irradiation there was a statistically significant shift in the proportion of EAC and E binding lymphocytes, with a decrease of T lymphocytes to 45% and a relative increase of B cells to 52%. The ability of lymphocytes to transform after in-vitro stimulation with P.H.A. was decreased in fifteen of nineteen patients tested. The data suggest that irradiation for mammary carcinoma leads to a long-lasting lymphopenia which mainly is a selective T-lymphocyte lymphopenia. These findings may be relevant for the development of distant metastases.

Downregulation of bactericidal peptides in enteric infections: A novel immune escape mechanism with bacterial DNA as a potential regulator

Antibacterial peptides are active defense components of innate immunity. Several studies confirm their importance at epithelial surfaces as immediate barrier effectors in preventing infection. Here we report that early in Shigella spp. infections, expression of the antibacterial peptides LL-37 and human β-defensin-1 is reduced or turned off. The downregulation is detected in biopsies from patients with bacillary dysenteries and in Shigella- infected cell cultures of epithelial and monocyte origin. This downregulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Analyses of bacterial molecules causing the downregulation indicate Shigella plasmid DNA as one mediator.

An Analysis of the Murine NK Cell as to Structure, Function and Biological Relevance

The immune system in higher animals has as a major distinguishing feature a high degree of complexity. The diversified machinery in this system can be considered to represent the gradual accumulation of preserved experiences during phylogeny where primitive as well as sophisticated reactions occur in parallel during the immune response. Interactions of complicated nature are known to occur within the system and the actual effector part (s) in causing the actual rejection of, e.g. an invading organism, is frequently difficult to dissect free from other contributing factors. The cellular part of the immune system can be divided in many ways. One approach is to talk about specific cells (most people would then mean lymphocytes of B and T type that effectuate their functions via their own, antigen-specific receptors) in contrast to nonspecific cells (here often granulocytes and monocytes-macrophages are linked together). Specific cells can function via selective production of their unique antigen-

T lymphocytes in collagen II-induced arthritis in mice: characterization of arthritogenic collagen II-specific T-cell lines and clones

Collagen type II‐spedfic long‐term cultured T helper cells, derived from the DBA/1 mouse, have been established and characterized. Clones from these T‐cell lines could he shown to recognize either species‐specific or species‐nonspecific determinants on the collagen type II molecule, including determinants on autologous mouse collagen. Induction of arthritis via transfer to both irradiated and normal syngeneic recipient mice was obtained with both collagen type II‐specific T‐cell lines and an autoreactive and collagen type II‐specific T‐cell clone. Fewer cells were needed to evoke arthritis in normal than in irradiated recipients. Cells from lines and the clone used for transfer were by immunocytochemistry shown to have T helper phenotype.

Antigen-Binding, Idiotypic T-Lymphocyte Receptors

Specific immune cognition and recognition are intrinsic capacities of mature, immunocompetent lymphocytes. B and T lymphocyte populations can both be shown to display this ability with high discriminatory power. At the single-cell level, however, both B and T lymphocytes express antigen-binding receptors with extreme restriction (Raff et al., 1973; Binz and Wigzell, 1975a). It would seem likely that in fact all receptors for antigen on a single B (Raff et al., 1973) or T (Binz and Wigzell, 1975a) lymphocyte express the same antigen-binding specificity. When T and B lymphocytes are compared with respect to their capacity to react against various epitopes, largely overlapping recognition spectra of antigenic specificities are found (Rajewsky and Mohr, 1974). Comparable mechanisms for the generation of diversity at the single-cell level of T and B cells would thus seem logical and economical. Whereas the biochemistry and underlying genetics of the B-cell receptors for antigen are by now relatively well understood, considerably less is known of the T-cell system.

Separation of Cells According to Surface Antigens by the Use of Antibody‐Coated Columns. Fractionation of Cells Carrying Immunoglobulins and Blood Group Antigen

It has been found possible to separate cells according to their surface anti gens by the use of antibody‐coated columns. High efficiency columns were made by double‐layer principles, first coating beads with antigen followed by antibody in excess. Such columns could be shown to contain a high amount of free antigen‐binding sites for the relevant antigens. Lymphoid cells were thus fractionated according to their surface concentration of immunoglobu lin and a highly selective retention of mouse B lymphocytes was observed when filtering spleen cells through an anti‐immunoglobulin column prepared according to the above procedure. No evidence of retention of mouse T lymphoid cells was observed in the same system. By the use of anti‐gamma‐2a immunoglobulin columns, it was found possible to deplete a population from memory cells potentially capable of synthesizing gamma‐2a antibodies. No evidence was found that columns prepared in the described manner would function through combining with receptors on lymphoid cells for antibody‐antigen complexes. By using anti‐A blood group columns, it was possible to selectively retain cells (erythrocytes or kidney cells) with A blood group anti gen on their surface. High‐avidity immune antibodies were found to be more efficient than ‘natural’ anti‐A antibodies in this test. No evidence was found of anti‐A antibodies being adsorbed on to the passing cells as tested by in vitro serological tests and tissue culture experiments. The applications of a technique for separating cells according to their surface antigens are con sidered obvious.

The role of IFN-γ in the outcome of chlamydial infection

Chlamydia are intracellular bacteria which infect many vertebrates, including humans. They cause a myriad of severe diseases, ranging from asymptomatic infection to pneumonia, blindness or infertility. IFN-gamma plays an important role in defense against acute infection and in the establishment of persistence. Chlamydia have evolved mechanisms to escape IFN-gamma functions. IFN-gamma-mediated effector mechanisms may involve effects on the metabolism of tryptophan or iron, on the inducible NO synthase (iNOS), on the secretion of chemokines and adhesion molecules or on the regulation of T-cell activities. IFN-gamma is secreted by the innate and the adaptive arms of the immune system. Within the former, Chlamydia-infected macrophages express IFN-gamma that in turn mediates resistance to infection. IFN-alpha/beta are pivotal for both IFN-gamma- and iNOS-mediated resistance to chlamydial infection in macrophages.

Lymphocyte mediated cytotoxicity in vitro. Induction and inhibition by humoral antibody and nature of effector cells.

During the past decade, experimental evidence has been brought forward in many laboratories that lymphocytes are capable of destroying appropriate target cells in vitro. These cytotoxic effects of lymphocytes have been assumed to reflect their effector role in tissue damaging immune responses such as occur in allograft rejection, tumor surveillance and certain autoimmune diseases (Perlmann & Holm 1969). Lymphocyte mediated cytotoxicity in vitro is complex and may involve a number of different pathways. The contention that a cytotoxic reaction is lymphocyte induced does not necessarily imply that the lymphocytes which start the reaction also are the cytotoxic effector cells, nor that the latter are lymphocytes at all. In addition, even in those instances in which there is evidence that both inducerand effector cells are lymphocytic, lymphocytes of different origin and function may participate in the cytotoxic reaction. Work with mice in alloimmune situations, involving the H2 system, has provided evidence that thymus derived lymphocytes (T-cells) are required for cytotoxicity (Blomgren et at. 1970, Cerottini et al. 1970a,b, Golstein et al. 1972a). In a few instances available data suggest that T-cells may be both inducerand effector cells (Golstein et al. I972a,b). Recent evidence also indicates that humoral antibodies may induce cytotoxicity of thymus-independent lymphoid cells (Harding et al. 1971, Van Boxel ct al.

Shared Idiotypic Determinants on B and T Lymphocytes Reactive Against the Same Antigenic Determinants

Normal rat lymphocyte populations house a high percentage of lymphocytes with idiotypic, antigen‐binding receptors for the major histocompatibility complex antigens of the rat. These receptors can be isolated from normal serum or lymphocyte supernatants. Idiotypic, antigen‐binding molecules released from normal Lewis lymphocytes were thus isolated using anti‐(Lewis anti‐DA) immuno‐adsorbents. Analysis by SDS polyacrylamide elertrophoresis using molecules labeled by external or internal means (125I or 3H) demonstrated that B lymphocytes produce molecules of ‘conventional’7S–8S IgM type. T‐lymphocyte‐derived molecules had a molecular weight of around 150,000 and consisted of two chains of similar size. Such single chains would succumb to proteolysis by normal serum factors to yield fragments in the size range of 30,000–40,000 daltons. All three groups of T‐cell‐derived molecules expressed both antigen‐binding and idiotypic markers. No evidence was obtained that any light chains are linked to the T‐receptor molecules. Serological analysis of the T‐cell molecules failed to prove the existence of any determinants of constant immunoglobulin type, nor did these molecules express antigenic markers of major histocompatibility complex types.