Induction of Interleukin 1α (IL-1α) and IL-1β mRNA Expression and Cellular IL-1 Production by Anti-HLA-DR Antibodies in Human Monocytes

We studied the role of HLA class II antigens in the regulation of interleukin 1 (IL-1) production in human monocytes. Monocytes were cultured with monoclonal anti-HLA-DR antibodies for 24 h after which cellular (i.e. intracellular and membrane-associated) IL-1 production, IL-1 secretion, and the expression of IL-1 alpha and IL-1 beta mRNA were determined. One of the anti-HLA-DR antibodies tested (anti-HLA-DR, Becton Dickinson) clearly induced IL-1 alpha and IL-1 beta mRNA expression and cellular IL-1 production. The other anti-HLA-DR antibody tested (OKIa1, Ortho) had no effect on IL-1 production. The stimulatory effect of anti-HLA-DR was enhanced by IFN-gamma in both fresh and aged monocytes. A synergistic effect by anti-HLA-DR and suboptimal doses of LPS (1 ng/ml) on both cellular IL-1 production and secretion was also demonstrated. The possibility of contaminating LPS causing the IL-1-inducing effect of anti-HLA-DR was excluded by the inability of polymyxin B to abolish the anti-HLA-DR-induced IL-1 production.     

Differential Induction of Membrane-Associated Interleukin 1 (IL-1) Expression and IL-1α and IL-1β Secretion by Lipopolysaccharide and Silica in Human Monocytes

Bacterial lipopolysaccharide (LPS) and silica dust are known to be effective inducers of interleukin 1 (IL-1) in human cultured monocytes. The data reported here show that although the levels of secreted IL-1 were equally high after in vitro stimulation with an optimal dose of LPS or silica, there were two clear differences: (i) the levels of membrane-associated IL-1 (as detected by the comitogenic effect of paraformaldehyde (PFA)-fixed cells or purified membrane fragments on murine thymocytes) were ca. five times higher after LPS stimulation than after silica stimulation, (ii) the secreted IL-1 after LPS stimulation was mainly of the pI 7 (IL-1 beta) type, while after silica stimulation there were equally high amounts of pI 7 and pI 5 (i.e. IL-1 alpha) forms. In both cases the IL-1 active molecules belonged to the 15 kDa class. These data show that the nature of the activating agent has a clear influence on the distribution of the biologically active IL-1 molecules. Moreover, the finding that after silica stimulation the amount of membrane-associated IL-1 (which was recently shown to be of the IL-1 alpha type) was low, while IL-1 alpha in the culture fluid was clearly elevated, suggests that the IL-1 alpha not attached to the cell membrane (or released from it) significantly contributes to the secreted IL-1 pool.     

Different Activation States of Human Lymphocytes after Antibody-Mediated Stimulation via CD3 and the α/β T-Cell Receptor

BMA031 is an IgG2b antibody directed towards the human alpha/beta T-cell receptor that is able to induce proliferation of peripheral blood mononuclear cells independent of antibody crosslinking. The proliferative response to BMA031 during the first 3 days of culture is usually of similar magnitude to that induced by the IgG2a CD3 antibody OKT3 but decreases quickly afterwards. Stimulation by BMA031 induces no measurable IL-2 release, very low expression of the IL-2 receptor, and does not trigger cytotoxic effector function. However, cross-linking of the antibody or addition of IL-2 leads to enhanced and prolonged proliferation, strong IL-2 receptor expression, and cytotoxic activity, features that are usually found after stimulation by the IgG2a CD3 antibody OKT3 in soluble form. The stimulatory effect of BMA031 cannot be diminished by IL-2 receptor blocking, whereas stimulation by OKT3 is strongly reduced. Moreover, proliferation induced by BMA031 has lower sensitivity to inhibition by ciclosporin than OKT3. From these results two major conclusions can be drawn: (1) an IL-2-independent way of activation may be important for the short-term proliferation of the T cells stimulated by BMA031 and (2) after stimulation by BMA031, cells reach a state of activation that is different from that induced by OKT3. These differences are most likely related to the different specificities of the antibodies, alpha/beta TcR versus CD3, suggesting that different activation signals are triggered via CD3 and via the alpha/beta TcR.     

Human NK Cells Expressing α4β1/β7 Adhere to VCAM-1 Without Preactivation

The authors demonstrate that resting CD56⁺/CD3⁻ NK cell adhesion to the endothelial VCAM-1 is over three-fold higher than CD56⁻/CD3 ⁺ T-cell adhesion. T-cell, but not NK-cell adhesion, to VCAM-1 is enhanced significantly by stimulation. The expression of VCAM-I receptor subunits α4 and β1 on both effector cells remains unchanged upon stimulation. A subpopulation of NK cells, as well as of T cells, was found to express β7, whose expression was not altered upon stimulation. The authors conclude that the adhesive properties of the same receptor structures on these distinct cell populations are regulated in a different manner, according to the specific functions of the effector cells of the immune system.     

Human α-lactalbumin and bovine β-lactoglobulin absorption in infants

We investigated gut permeability to human α-lactalbumin (ALA) and bovine β-lactoglobulin (BLG) in 20 infants from birth to 8 months or until weaning, before which they were on a strictly cow's-milk-free diet. We measured the proteins with a sensitive, solid-phase, double-sandwich immunofluorometric assay. Median (range) levels of serum ALA on days 3-4 after birth, and at 1 and 2 months of age were 31 (12-225), 6 (0–55), and 2 (0–16) μg/1 serum per g ALA given per kg body weight, respectively. At 3, 5, and 8 months of age, only trace amounts of ALA were found. One week after weaning, serum BLG was found in 5/13 infants (38%) and at 2 weeks in 3/14 infants (21°0), with median concentrations of 7 and 4 μg/1 serum per g BLG given per kg body weight, respectively. No ALA could be detected in any of these samples. In absorption of ALA, the four infants who had allergic symptoms did not differ from those without symptoms. Thus, systemic absorption of ALA and BLG does occur in infants. Absorption of ALA is greatest after birth, when 3 × 10⁴ (median) of the given antigens are absorbed, but absorption decreases rapidly. The gut may often be transiently permeable to BLG when cow's-milk-based formula is started.     

Emergence of α5β1 fibronectin- and αvβ3 vitronectin-receptor expression in melanocytic tumour progression

Cell adhesion is crucial in the process of tumour progression. As integrins are important receptor molecules involved in cell adhesion, we studied the distribution of the α1-6, αv, αIIb, β1, β3, and β4 integrin subunits in tissue sections of common naevocellular naevi ( n =22), dysplastic naevi (16), thin (24) and thick primary cutaneous melanomas (28), and melanoma metastases (25). We found correlated expression of α1/α2, of α4/α5/β3, and of α6/β4. Decrease of α6 and β4, and increase of α4 and αv were found to be correlated with melanoma progression. Furthermore, expression of α5 and β3 was detected only in primary melanoma and melanoma metastasis. Our findings indicate that during melanoma progression alterations in integrin expression occur, the most striking being emergence of α5β1 fibronectin and αvβ3 vitonectin receptor.     

Cyclic Adenosine Monophosphate Decreases the Secretion, but not the Cell-Associated Levels, of Interleukin-1β in Lipopolysaccharide-Activated Human Monocytes

Interleukin-1 beta (IL-1 beta) is a cytokine produced mainly by activated monocytes though the mechanism by which it is released is still unknown. Elevation of intracellular cyclic adenosine monophosphate (cAMP) is considered an important down-regulative signal in the production of IL-1 beta in lipopolysaccharide (LPS)-induced monocytes. In this study we show that in LPS-activated human monocytes, elevated cAMP concentrations (induced by either prostaglandin E2, forskolin or dibutyrylcyclic AMP) affected specifically secretion of IL-1 beta; the amount of secreted IL-1 beta was clearly reduced whereas the cell-associated level remained unchanged. TNF-alpha, a normal secretory protein, was used as a control. Cyclic AMP also inhibited TNF production by monocytes, but the decrease was of the same magnitude in the extracellular and intracellular compartments. Thus, the down-regulative effect of cAMP on the production of these monokines is clearly different.     

Evolutionarily Conserved Function of CD28 in αβ T Cell Activation

The functional role of the chicken homologue of CD28 was studied. It is expressed on all thymocytes, and both Vβ1- and Vβ2-family expressing peripheral αβ T cells. Peripheral γβ T cells are CD28-negative. Monoclonal antibody against CD28 had a costimulatory effect on T cells stimulated by phorbol myristate acetate (PMA), concanavalin A or MoAb against TCR. Vβl and Vβ2 expressing cells responded equally well to stimulation with anti-CD28 in combination with PMA. These responses were resistant to cyclosporin A, but inhibited by herbimycin A, suggesting that CD28 employs a signalling pathway at least partly distinct from that triggered by TCR/CD3. These data indicate a striking conservation of the costimulatory function of CD28 and emphasize the importance of this costimulatory pathway.     

Anti-GD 3 Antibodies are Potent Activators of Human γ/δ and α/β Positive T Cells

The ganglioside GD3 has a variety of biological functions. These include stimulatory effects is on proliferation, natural killer activity and cytokine production by freshly isolated peripheral T cells. In this study we have characterized anti-GD3 antibody (MoAb Z21) mediated effects on T cell clones. Our data indicate that α/β TCR CD4⁺ and CD8⁺ as well as γ/δ TCR positive T cells can be stimulated resulting in proliferation and cytokine production. This effect could be blocked by cyclosporin A and did not involve the LFA-3 or CD4 molecule. Apart from IFN-γ and IL-2 production by T helper I and T helper 0 cells we have observed production of IL-4 and IL-10 by T helper 2 cells indicating that the GD3 molecule is not a marker for a certain functional T cell subset. In contrast to anti-CD3 mediated activation, the responsiveness of T cells to stimulation via GD3 was dependent on the cell surface expression of the molecule and could be enhanced by costimulation via CD2, CD3, CD26 or CD28. In addition, anti-GD3 antibodies delivered a potent costimulatory signal for antigen-induced proliferation of CD4⁺ T lymphocytes. In summary, our experiments illuminate the mechanisms of anti-GD3 antibody induced T cell activation.     

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Gamma/delta TCR bearing T lymphocytes represent a T-cell subset whose functional relevance remains unclear. Nevertheless these T cells may play a role in the early immune reponse against bacteria. Until now the regulatory mechanisms on this response have not been investigated. The study described here evaluated the immunoregulatory effects of Interleukin-10 on γ/δ and α/β TCR-positive T-cell clones and freshly isolated peripheral-blood mononuclear cells (PBMC). IL-10 has been shown previously to inhibit lectin and antigen-induced proliferation and cytokine production by α/β T cells. The results outlined below show that rhIL-10 strongly inhibits lectin-induced production of IFN-γ, TNF-α. IL-2, and to a lesser degree proliferation and IL-4 production of both T-cell subsets. As IL-10 did not inhibit proliferation but at the same time strongly suppressed cytokine production in various experiments, the hypothesis that it could function as a growth factor for human T cells as has been described for murine thymoeytes was tested. The data demonstrate that, although the γ/δ T-cell clones tested do not produce IL-10 they can use it as a growth factor in combination with IL-2, IL-4 or alone. Furthermore, IL-10 has the same properties on human α/β T-cell clones and PBMC. In summary, it is shown that IL-10 has pleiotropic effects on γ/δ and α/β TCR⁺ T cells by inhibiting lectin-induced cytokine production and by acting as a growth factor for these cells alone or in combination with IL-2 or IL-4.     

Bio-histochemical aspects of integrins (α2β1, α6β1) in invasive mammary carcinomas: An immunohistochemical study

Immunohistochemical expression of integrins was examined in 39 human invasive mammary carcinomas, of which 34.2% and 43.6% expressed integrins α2β1 and α6β1, respectively. Immuno-electron microscopy clearly demonstrated that the integrins were in the cell membrane of the carcinoma cells. Similar expression of integrin α2β1 or α6β1 in both the intraductal component and invasive portion of the same tumor was seen in 76.9% and 85.7% of cases, respectively. This suggested that invasive carcinoma cells retained their integrin expression after invasion through the basement membrane. Reciprocal expression of integrins α2β1 and α6β1 was seen in 20 cases. Expression of α2β1 was seen significantly less frequently in scirrhous carcinoma than in the more differentiated papillotubular or solid tubular carcinoma (Chi-squared test, P < 0.05). Intraductal components of carcinoma were present more frequently in cases expressing integrin α2β1 than in those that were negative. This suggests the potential usefulness of integrins as clinical parameters in the surgical treatment of mammary carcinomas, since recent trials of conservative treatment for mammary carcinoma have focused on the intraductal spread of the tumor cells.     

T Lymphocytes in Human Gut Epithelium Preferentially Express the α/β Antigen Receptor and are often CD45/UCHL1-Positive

A revived interest in intraepithelial lymphocytes (IEL) has been elicited by several recent reports suggesting that murine and avian intestinal epithelium contains mainly CD3+CD8+ cells expressing the gamma/delta T-cell receptor (TcR) for antigen; this contrasts with systemically distributed T cells which preferentially employ the TcR alpha/beta. An anatomical dichotomy in the distribution of these two T-cell lineages has hence been proposed. Here we report that this concept does not hold true in man. In situ studies with monoclonal TcR-framework antibodies showed that most (70-90%) human intestinal IEL (which are mainly CD3+CD8+) expressed TcR alpha/beta. Moreover, almost half of the intraepithelial CD3+ cells were positive for the smallest (180 kDa) CD45 molecule (UCHL1); this probably reflected that they are antigen-primed and thus represent traditional CD3+CD8+ alpha/beta+ memory T cells.     

Repertoire of the αβ T-cell receptor in the intestine

Summary:  The majority of T cells in the human and mouse intestine express the T-cell receptor (TCR) as an αβ heterodimer on their cell surface. As the major recognition element of antigens in the context of major histocompatibility complex-derived proteins, an examination of the structure of the αβTCR in intestines has provided significant insights into the potential function of these cells and the major determinants that drive their selection. Studies in the human intestine have shown that the repertoires of intraepithelial lymphocytes (IELs), and likely lamina propria lymphocytes, are polyclonal before and shortly after birth, with the repertoire becoming oligoclonal in adults. Similarly, in adult mice the repertoire is oligoclonal, while in the newborn it is polyclonal. Investigations in mice have shown that some T cells may evade thymic selection. The population size and oligoclonality of IELs is influenced by the microbial content of the luminal microenvironment. This microenvironment probably directly determines the TCR repertoire. Studies in human inflammatory bowel disease (IBD) indicate that inflammation further skews the TCR repertoire. We speculate that dominant antigens associated with the pathogenesis of IBD are responsible for such skewing and that identifying the antigenic drivers may shed light on the environmental factors that trigger or potentiate human IBD.     

Dermatitis herpetiformis and celiac disease are both primarily associated with the HLA-DQ (α1*0501, (β1*02) or the HLA-DQ (α1*03, (β1*0302) heterodimers

HLA-DRB1, -DQA1, and -DQB1 genomic typing of 50 patients with dermatitis herpetiformis and of 290 healthy blood donors was performed. Genes encoding the DQ (α1*0501, (β1*02) heterodimer were carried by 43 (86%) of the patients and 72 (25%) of the controls. Of die remaining seven patients six (12% of all the patients) carried genes encoding the DQ (α1*03, β1*0302) heterodimer. These HLA associations are very similar to those observed in patients with celiac disease. We thus conclude that dermatitis herpetiformis and celiac disease are associated to the very same HLA-DQαβ heterodimers.     

Comparison of Biological and Immunological Activities of Human Monocyte-Derived Interleukin 1β and Human Recombinant Interleukin 1β

Recombinant human interleukin 1 beta (rhIL-1 beta) and supernatants of Escherichia coli lipopolysaccharides-stimulated human monocyte (Mo) cultures, containing native human IL-1 beta (nhIL-1 beta), demonstrate significant differences when tested in the mouse co-stimulatory thymocyte (lymphocyte activating factor [LAF]) assay. The aims of the present study were to investigate this characteristic difference between rhIL-1 beta and Mo culture supernatants (Mo supernatants), and to compare the biological and the immunological activity of preparations of rhIL-1 beta and nhIL-1 beta during each step of an identical purification procedure. The biological activity of rhIL-1 beta/nhIL-1 beta preparations was characterized by the use of the LAF assay and the rat islet insulin release assay. An IL-1 beta enzyme-linked immunosorbent assay (ELISA) was established in order to compare the biological and immunological responses of the IL-1 beta preparations. We report that the significant difference between rhIL-1 beta and supernatants of Mo cultures, which was only demonstrable in the LAF assay, is due to the presence of interleukin 6 (IL-6) in the Mo supernatants. We describe a simple cation exchange chromatography separating nhIL-1 beta and IL-6 of Mo supernatants. The highly purified rhIL-1 beta possessing the correct amino-terminal sequence and nhIL-1 beta have identical biological and immunological activities demonstrating a specific biological activity (SBA) of 3 x 10(2) U/ng IL-1 beta. Thus, we have no indications of secondary or tertiary structural differences between rhIL-1 beta and purified nhIL-1 beta. In contrast, both in the LAF assay and in the rat islet insulin release assay the SBA of an amino-extended rhIL-1 beta form, Met-Glu-Ala-Glu-rhIL-1 beta, was only 1-2% of the SBA of rhIL-1 beta, suggesting that structural changes were introduced into the molecule by the amino-terminal extension. In the present study we have demonstrated that systematic combined testing of IL-1 beta preparations in two different biological assays and an immunological assay is useful for the characterization and comparison of the activity of recombinant and native IL-1 beta preparations purified by the use of exactly the same procedures.