Lennart Lindbom

Phagocyte partnership during the onset and resolution of inflammation.

Neutrophils, monocytes and macrophages are closely related phagocytic cells that cooperate during the onset, progression and resolution of inflammation. This Review highlights the mechanisms involved in the intimate partnership of phagocytes during each progressive phase of the inflammatory response. We describe how tissue-resident macrophages recognize tissue damage to promote the recruitment of neutrophils and the mechanisms by which infiltrating neutrophils can then promote monocyte recruitment. Furthermore, we discuss the phagocyte-derived signals that abrogate neutrophil recruitment and how the uptake of apoptotic neutrophils by macrophages leads to termination of the inflammatory response. Finally, we highlight the potential therapeutic relevance of these interactions.

Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability.

Polymorphonuclear leukocyte infiltration into tissues in host defense and inflammatory disease causes increased vascular permeability and edema formation through unknown mechanisms. Here, we report the involvement of a paracrine mechanism in neutrophil-evoked alteration in endothelial barrier function. We show that upon neutrophil adhesion to the endothelial lining, leukocytic β2 integrin signaling triggers the release of neutrophil-borne heparin-binding protein (HBP), also known as CAP37/azurocidin, a member of the serprocidin family of neutrophil cationic proteins. HBP induced Ca++-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial-cell monolayers in vitro, and increased macromolecular efflux in microvessels in vivo. Moreover, selective inactivation of HBP prevented the neutrophils from inducing endothelial hyperpermeability. Our data suggest a fundamental role of neutrophil-derived HBP in the vascular response to neutrophil trafficking in inflammation. Targeting this molecule in inflammatory disease conditions offers a new strategy for prevention of endothelial barrier dysfunction caused by misdirected leukocyte activation.

M PROTEIN, A CLASSICAL BACTERIAL VIRULENCE DETERMINANT, FORMS COMPLEXES WITH FIBRINOGEN THAT INDUCE VASCULAR LEAKAGE

Increased vascular permeability is a key feature of inflammatory conditions. In severe infections, leakage of plasma from the vasculature induces a life-threatening hypotension. Streptococcus pyogenes, a major human bacterial pathogen, causes a toxic shock syndrome (STSS) characterized by excessive plasma leakage and multi-organ failure. Here we find that M protein, released from the streptococcal surface, forms complexes with fibrinogen, which by binding to beta2 integrins of neutrophils, activate these cells. As a result, neutrophils release heparin binding protein, an inflammatory mediator inducing vascular leakage. In mice, injection of M protein or subcutaneous infection with S. pyogenes causes severe pulmonary damage characterized by leakage of plasma and blood cells. These lesions were prevented by treatment with a beta2 integrin antagonist. In addition, M protein/fibrinogen complexes were identified in tissue biopsies from a patient with necrotizing fasciitis and STSS, further underlining the pathogenic significance of such complexes in severe streptococcal infections.

Neutrophil secretion products pave the way for inflammatory monocytes

The leukocyte response in inflammation is characterized by an initial recruitment of polymorphonuclear leukocytes (PMN) preceding a second wave of monocytes to the site of injury or infection. In the mouse, 2 populations of monocytes have been identified, Gr1(-)CCR2(-)CX3CR1(hi) resident monocytes and Gr1(+)CCR2(+)CX3CR1(lo) inflammatory monocytes. Here, intravital microscopy of the musculus cremaster and a subcutaneous air pouch model were used to investigate a possible link between PMN extravasation and the subsequent emigration of inflammatory monocytes in response to local stimulation with PAF. In mice that were made neutropenic by injection of a PMN-depleting antibody, the extravasation of inflammatory monocytes, but not resident monocytes, was markedly reduced compared with mice with intact white blood cell count but was restored by local treatment with secretion of activated PMN. Components of the PMN secretion were found to directly activate inflammatory monocytes and further examination revealed PMN-derived LL-37 and heparin-binding protein (HBP/CAP37/azurocidin) as primary mediators of the recruitment of inflammatory monocytes via activation of formyl-peptide receptors. These data show that LL-37 and HBP specifically stimulate mobilization of inflammatory monocytes. This cellular cross-talk functionally results in enhanced cytokine levels and increased bacterial clearance, thus boosting the early immune response.

Leukocyte‐Cell Adhesion: A Molecular Process Fundamental in Leukocyte Physiology

Leukocyte-cell adhesion is a form of physical contact characterized by fast (firm) stickiness between the cells. To analyze the biology and molecular basis of this process, an adhesion-specific assay was developed: the phorbol ester-induced aggregation of human lymphocytes. This rapid and antigen-independent intercellular adhesion requires cellular metabolism, an intact cytoskeleton and extracellular divalent cations, and is mediated by preformed cell-surface proteins referred to as CAMs. Phorbol ester also induces aggregation of monocytes and granulocytes, as well as adhesion of T lymphocytes to either B cells or monocytes and of the leukocytes to vascular endothelial cells. By using the adhesion-specific assay and blocking monoclonal antibodies, several CAMs have been identified, namely the Leu-CAM family (CD11a-c/CD18) and ICAM-1 (CD54). The Leu-CAM family is composed of Leu-CAMa (CD11a/CD18), Leu-CAMb (CD11b/CD18) and Leu-CAMc (CD11c/CD18), three glycoprotein heterodimers made of a common beta-chain and distinct alpha-chains. ICAM-1 is an adhesive ligand for Leu-CAMa. Expression and use of the various CAMs is selective in different types of leukocytes. The Leu-CAMs have been purified and partially characterized. CD18, whose gene is on human chromosome 21, contains 5-6 N-linked complex-type oligosaccharides, and CD11 binds Ca++. Another adhesion pathway is mediated by CD2 and CD58. CD2, a glycoprotein selectively expressed by T cells, is a receptor for CD58, a cell-surface adhesive ligand with broad tissue distribution. Antibodies to the latter CAMs do not block the phorbol ester-induced lymphocyte aggregation. Adhesion is involved in a large variety of leukocyte functions. Anti-Leu-CAM antibodies block induction of IL-2 production and lymphocyte proliferation. Lymphocyte-mediated cytotoxicity is also inhibited. Endogenous NK and LAK cells use Leu-CAMs, ICAM-1 and CD2, and sometimes RGD receptors, to bind and kill tumor cells. Endogenous compounds such as H2O2 and LTB4 also induce Leu-CAM-dependent adhesion in monocytoid cells and granulocytes, respectively, and degranulation of the latter cells is enhanced by the adhesion process. Homologous CAMs have been identified in rabbit and mouse. In in vivo studies in the former species, anti-Leu-CAM antibodies block adhesion of leukocytes to vascular endothelium and thereby their migration into extravascular tissues. The antibodies thus inhibit granulocyte accumulation and plasma leakage in inflammatory lesions, and induce lympho- and granulocytosis, indicating that cell-adhesion contributes to the distribution of leukocytes in the body.(ABSTRACT TRUNCATED AT 400 WORDS)

Neutrophil primary granule proteins HBP and HNP1–3 boost bacterial phagocytosis by human and murine macrophages

In acute inflammation, infiltrating polymorphonuclear leukocytes (also known as PMNs) release preformed granule proteins having multitudinous effects on the surrounding environment. Here we present what we believe to be a novel role for PMN-derived proteins in bacterial phagocytosis by both human and murine macrophages. Exposure of macrophages to PMN secretion markedly enhanced phagocytosis of IgG-opsonized Staphylococcus aureus both in vitro and in murine models in vivo. PMN secretion activated macrophages, resulting in upregulation of the Fcgamma receptors CD32 and CD64, which then mediated the enhanced phagocytosis of IgG-opsonized bacteria. The phagocytosis-stimulating activity within the PMN secretion was found to be due to proteins released from PMN primary granules; thorough investigation revealed heparin-binding protein (HBP) and human neutrophil peptides 1-3 (HNP1-3) as the mediators of the macrophage response to PMN secretion. The use of blocking antibodies and knockout mice revealed that HBP acts via beta2 integrins, but the receptor for HNP1-3 remained unclear. Mechanistically, HBP and HNP1-3 triggered macrophage release of TNF-alpha and IFN-gamma, which acted in an autocrine loop to enhance expression of CD32 and CD64 and thereby enhance phagocytosis. Thus, we attribute what may be a novel role for PMN granule proteins in regulating the immune response to bacterial infections.

Neutrophil granule proteins tune monocytic cell function

Polymorphonuclear leukocytes (PMNs) release the contents of granules during their migration to inflammatory sites. On liberation from the first leukocyte to enter injured tissue, the granule proteins play a central role in the early inflammatory response. In particular, mononuclear phagocytes interact intimately with PMNs and their secretion products. PMN granule proteins enhance the adhesion of monocytes to the endothelium and stimulate subsequent extravasation of inflammatory monocytes. At the site of inflammation, PMN granule proteins activate macrophages to produce and release cytokines and to phagocytose IgG-opsonized bacteria. Furthermore, by direct cell-cell contacts, PMNs activate monocyte-derived dendritic cells, thereby enhancing antigen presentation. Efforts in this field might lead to the development of drugs for specific modulation of innate immune functions.

Streptococcal M Protein: A Multipotent and Powerful Inducer of Inflammation

Severe infections with Streptococcus pyogenes, an important human pathogen, are associated with massive inflammatory reactions in the human host. Here we show that streptococcal M protein interacts with TLR2 on human peripheral blood monocytes. As a consequence, monocytes express the cytokines IL-6, IL-1β, and TNF-α. This response is significantly increased in the presence of neutrophil-derived heparin-binding protein (HBP), which costimulates monocytes by interacting with CD11/CD18. Analysis of tissue biopsies from patients with necrotizing fasciitis revealed recruitment of neutrophils and monocytes to the infectious site, combined with the release of HBP. The results show that M protein, in synergy with HBP, evokes an inflammatory response that may contribute to the profound pathophysiological consequences seen in severe streptococcal infections.

Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins

Leukocyte infiltration in atherosclerosis has been extensively investigated by using histological techniques on fixed tissues. In this study, intravital microscopic observations of leukocyte recruitment in the aorta of atherosclerotic mice were performed. Interactions between leukocytes and atherosclerotic endothelium were highly transient, thereby limiting the ability for rolling leukocytes to firmly adhere. Leukocyte rolling was abolished by function inhibition of P‐selectin (P<0.001, n=8), whereas antibody blockage of E‐selectin (n= 10) decreased rolling leukocyte flux to 51 ± 9.9% (mean±SE, P<0.01) and increased leukocyte rolling velocity to 162 ± 18% (P<0.01) of pretreatment values. Notably, function inhibition of the integrin α4 subunit (n=5) had no effect on rolling flux (107±25%, P=0.782) or rolling velocity (89±6.1%, P=0.147), despite endothelial expression of vascular cell adhesion molecule 1 (VCAM‐1). Leukocytes interacting with atherosclerotic endothelium were predominantly neutrophils, because treatment with antineutrophil serum decreased rolling and neutrophil counts in peripheral blood to the same extent. In conclusion, we present the first direct observations of atherosclerosis in vivo.We show that transient dynamics of leukocyte‐endothelium interactions are important regulators of arterial leukocyte recruitment and that leukocyte rolling in atherosclerosis is critically dependent on the endothelial selectins. This experimental technique and the data presented introduce a novel perspective for the study of pathophysiological events involved in large‐vessel disease.—Eriksson, E. E., Xie, X., Werr, J., Thoren, P., Lindbom, L. Direct viewing of atherosclerosis in vivo: plaque invasion by leukocytes is initiated by the endothelial selectins. FASEB J. 15, 1149‐1157 (2001)

Distinct Infiltration of Neutrophils in Lesion Shoulders in ApoE−/− Mice

Inflammation and activation of immune cells are key mechanisms in the development of atherosclerosis. Previous data indicate important roles for monocytes and T-lymphocytes in lesions. However, recent data suggest that neutrophils also may be of importance in atherogenesis. Here, we use apolipoprotein E (ApoE)-deficient mice with fluorescent neutrophils and monocytes (ApoE(-/-)/Lys(EGFP/EGFP) mice) to specifically study neutrophil presence and recruitment in atherosclerotic lesions. We show by flow cytometry and confocal microscopy that neutrophils make up for 1.8% of CD45(+) leukocytes in the aortic wall of ApoE(-/-)/Lys(EGFP/EGFP) mice and that their contribution relative to monocyte/macrophages within lesions is approximately 1:3. However, neutrophils accumulate at sites of monocyte high density, preferentially in shoulder regions of lesions, and may even outnumber monocyte/macrophages in these areas. Furthermore, intravital microscopy established that a majority of leukocytes interacting with endothelium on lesion shoulders are neutrophils, suggesting a significant recruitment of these cells to plaque. These data demonstrate neutrophilic granulocytes as a major cellular component of atherosclerotic lesions in ApoE(-/-) mice and call for further study on the roles of these cells in atherogenesis.