**3. Histopathology**

Pathological expressions of vasculitis are not usually specific for a particular diagnostic catego‐ ry of vasculitis. The primary pathology in the majority of pulmonary-renal syndrome is inflammation and necrosis of vessel wall classified according to the Chapel Hill Consensus Conference classification [3] as medium/small vessel vasculitis. Generally, inflammation affects arterioles, capillaries and venules with two basic types of necrosis – fibrinoid and granuloma‐ tous. In the very early stage of inflammation there is a massive influx of polymorphonuclear leukocytes and monocytes accumulating in the capillary space. Later the condition is accompa‐ niedbyproteinacousexudate.Damageofthecapillariesanddisruptionofbasalmembraneswith leakageof erythrocytes is followedbyaninfluxofmacrophages.Fibrinoiddepositions cause the formation of extracapillary (crescent) cell proliferation in the glomerulus [11]. Changes in lungs give rise to isolated necrotic pulmonary capillaritis where damaged red blood cells migrate directly into alveolar tissue resulting in alveolar haemorrhage [12].

Jennette et al. [13] described pathologic features of different necrotizing vasculitis types indicating various pathogenic mechanisms causing the injury. In anti-GBM disease or immune complex disease the pathogenic complexes between antibodies and antigens are located exclusively or predominantly in vessel walls. In patients with ANCA vasculitis, these autoantibodies are in the interstitial fluid and also in the blood. ANCA activate neutrophils and monocytes in blood vessels, as well as in interstitial tissue. Activation in the vessels causes necrotizing vasculitis and activation in the tissues necrotizing tissue inflammation. Clinical association of dominating organs in pulmonary-renal syndrome is shown in table 1.


**Table 1.** Approximate frequency of organ system involvement (%)

Interstitial inflammation is accompanied by capillary trombosis, disintegration of blood vessel wall, loss of integrity of tissue structures, epithelial cell hyperplasia, accumulation of red cells (and later hemosiderin), depositions of proteins (immunoglobulins, immune-complex proteins), interstitial fibrosis and atrophy. The actual picture is dependent on different organ structure of lungs and kidneys, as well as on primary stimulus.

Presence of specific granulomatous inflammation is typical for granulomatosis with polyangiitis (GPA, formerly Wegener´s granulomatosis) described and defined by Wegener in 1939 [14] and Former in 1950 [15] respectively. Morphological detected recruitment of inflammatory cells, as well as immune competent cells (T-cells, B, cells, macrophages) and sometimes also giant-cells are confirmed by immunostaining of autoantibodies, detection of cytokine release and oxygen-free radical formation.

Pulmonary-renal syndrome has a wide spectrum of organ histopathological changes. Severe renal vascular damage can be accomapnied by absent or mild pulmonary changes. On the other hand severe pulmonary injury can be followed by mild renal destruction or normal renal histology.

#### **3.1. Renal pathology**

remain poorly understood. Although the primary events that initiate this process remain largely unknown, recent investigations have brought us closer to understanding some of the critical pathways involved in disease and provided a rationale for the study of novel

The first mention on pulmonary-renal syndrome is dated to year 1919 when the "father of viral pathology" in the United States Dr. Ernest William Goodpasture (1886 – 1960) published his work "The Significance of Certain Pulmonary Lesions in Relation to the Etiology of Influenza in American Journal of the Medical Sciences [5]. He described two cases from more than fifty autopsies where in patients dying in the great flu pandemic (Spanish flu) no bacterial etiology was confirmed. In one of the two patients massive alveolar hemorrhage and fulminant glomerulonephritis were present. It was never discovered what underlying disease other than influenza this patient may have had. Human influenza virus was described fourteen years later by Laidlaw and coworkers [6]. In 1958 two Australian scientists Stanton and Tange in the discussion of their paper analyzed Goodpasture´s old finding and associated pulmonary hemorrhage with glomerulonephritis with the name of Dr. Goodpasture, as Goodpasture syndrome [7]. According to the biography written by Collins (2010), E. Goodpasture did not approve the association of his name with this syndrome [8]. In 1967 the discovery of anti-GBM

At present pulmonary-renal syndrome broadened the family of diseases, where diffuse alveolar hemorrhage and immune crescent glomerulonephritis are participating. Therefore, it is not possible to say only glomerular basement membrane (Goodpasture) disease means pulmonary-renal syndrome. The term pulmonary-renal syndrome should be associated with

Pathological expressions of vasculitis are not usually specific for a particular diagnostic catego‐ ry of vasculitis. The primary pathology in the majority of pulmonary-renal syndrome is inflammation and necrosis of vessel wall classified according to the Chapel Hill Consensus Conference classification [3] as medium/small vessel vasculitis. Generally, inflammation affects arterioles, capillaries and venules with two basic types of necrosis – fibrinoid and granuloma‐ tous. In the very early stage of inflammation there is a massive influx of polymorphonuclear leukocytes and monocytes accumulating in the capillary space. Later the condition is accompa‐ niedbyproteinacousexudate.Damageofthecapillariesanddisruptionofbasalmembraneswith leakageof erythrocytes is followedbyaninfluxofmacrophages.Fibrinoiddepositions cause the formation of extracapillary (crescent) cell proliferation in the glomerulus [11]. Changes in lungs give rise to isolated necrotic pulmonary capillaritis where damaged red blood cells migrate

**2. Historical sense of the term "pulmonary - renal syndrome"**

antibodies were associated with Goodpasture nephritis [9].

the term pulmonary-renal vasculitic syndromes [10].

directly into alveolar tissue resulting in alveolar haemorrhage [12].

therapeutic agents [4].

74 Updates in the Diagnosis and Treatment of Vasculitis

**3. Histopathology**

Renal pathology is expressed by various forms of glomerulonephritis (anti-GBM, immunocom‐ plex, necrotizing pauci-immune). In rare cases sequential development supposed to be of pathogenic importance: injury caused by ANCA may uncover the Goodpasture antigen. The concept that only one antigen may trigger another one requires further support [16].

Immunohistochemical classification of renal capillary vasculitides [17] based on renal biopsies is characterized by the presence of:


#### **3.** pauci-immune ( only circulating ANCA) – type III crescentic GN

#### **3.2. Pulmonary pathology**

The underlying pulmonary lesions (necrotic pulmonary capillaritis) are clinically expressed as diffuse alveolar haemorrhage. Disruption and degradation of the pulmonary capillary wall and interstitial matrix results in vessel wall destruction and necrosis. Cordier and Cottin [18] found capillaritis as the most common pathological finding (60%) in ANCA-related vasculitides with pulmonary complications. However, capillaritis was not observed in all patients. It is likely that vessel inflammations may be overlooked in some cases if not specifically searched [19].

endothelial cells and so could expose components of the alveolar basement membrane to cells of the immune system, initiating an immune response [21]. The induction of vasculitis seems multifactorial, with interplay of environmental factors and genetic predisposition creating the

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Newexperimentaldatahaveemergedinrecentyearsfocusingontheinteractiveeffectsofkidney and lung dysfunction, and these studies have highlighted the pathophysiological importance of proinflammatory and other immunologic pathways as well as the complex nature of interor‐ gancrosstalk.Becausepulmonaryandrenaldysfunctionfrequentlycoexist,the effectsoffailure of either organ are particularly relevant to the function of the other. Evidence suggests that deleteriouskidney-lunginteractionsor crosstalkrise,atleastinpart,duetothelossofthenormal balanceofimmune,inflammatoryandsolublemediatormetabolism.Theseprocessesoccurafter severe insults and cause induction of organ injury [23]. Organ crosstalk is a consequence of both directlossofnormalfunctionandinflammatorydysregulationresultingfrombothorganfailure. Cellular(e.g.neutrophils) aswell as solublemediators (cytokines) contribute tothe inflammato‐

Interactions between inflammatory cells and damaged endothelium end in vessel inflammation (vasculitis), the major manifestation of all clinical entities. Cytokines, chemokines (such as IL-8) complement components, circulating immune complexes, and antibodies can be primary determinants of initiators of endothelial cell damage. When focusing on two basic clinical representatives – Goodpasture disease and granulomatosis with polyangiitis, damage of the vessel wall of glomeruli and alveolar capillaries are caused by antigen-stimulated white blood cells, anti-GBM antibodies and ANCA, respectively. In anti-GBM and ANCA vasculitis, the pathologic finding of focal, lytic necrotizing injury suggests highly effective local activation of neutrophils and monocytes with release of oxidants and

Concurrent ANCA and anti-GBM antibody production can be seen in selected patients, but reasonable explanation is unknown. It is possible that ANCA-related proteases damage or expose the nephritogenic epitopes in cr3 (IV) collagen in GBM, and this in turn leads to anti-GBM antibody production. It is unlikely that the crossreactivity between p-ANCA and anti-GBM antibodies is derived from the same autoantibody repertoire, because there does not appear to be a structural relationship between c-ANCA and a3 (IV) NC I collagen [25]. It is currently unclear whether there is any structural cross-reactivity between c-ANCA and anti-

Immunological mechanisms involved in initiation and prolongation of inflammatory state could be divided to two groups: cellular mechanisms (activity of immune-competent cells) and

environment for development of disease [22].

ry dysregulation under these circumstances [24].

proteases that are neutralized beyond the site of injury [13].

**4.3. Immunology of inflammation**

GBM antibodies [26].

**4.4. Cellular and humoral mechanisms**

**4.2. Kidney-lung crosstalk**

According to recommendations of Jennette and Falk [20] an accurate precise clinical diagnosis usually requires the integration of many different types of data, including clinical signs and symptoms, associated diseases, histological pattern of inflammation (eg, granulomatous versus necrotizing), immunopathological features (e.g. presence and composition of vascular immunoglobulin deposits), and serological findings (cryoglobulins, hypocomplementemia, hepatitis B antibodies, hepatitis C antibodies, ANCAs, anti–GBM antibodies, ANA). Specific diagnosis of a vasculitis is very important because the prognosis and appropriate therapy vary substantially among different types of vasculitis. Many attempts to re-evaluate and to refine the present nomenclature of vasculitis were done, however the complexity of vasculitic syndromes, as well as of pulmonary-renal syndrome complicates not only the estimation of appropriate diagnosis, differential diagnosis but also hampers effective treatment.

### **4. Immunopathogenesis**

Potentially accepted immunopathological mechanisms of pulmonary-renal syndrome involve antiglomerular basement membrane antibodies, antineutrophil cytoplasm antibodies, generation of immunocomplexes, activation of complement and haemocoagulation. Due to different pathogenesis of pulmonary-renal syndrome in various clinical diagnoses, common immunopathological features (except systemic inflammation) could not be specified. As seen in most inflammatory diseases, the systemic response to insult may be as important as the initial stimulus.

#### **4.1. Genetical and environmental influence**

The current understanding of autoimmune disorders suggests that some environmental factors initiate disease in a genetically susceptible individual. The importance of genetic factors, especially the genes of major histocompatibility complex has been increasingly recognized in determining susceptibility to autoimmune diseases. Because of the low incidence of all nosological entities of pulmonary-renal syndrome, it is not possible to examine the inheritance of genes within affected families predisposing to this disease.

In pulmonary-renal syndrome, as in most autoimmune disorders, the precise initiating events are not known. Exposure to hydrocarbons is known to cause damage to pulmonary/renal endothelial cells and so could expose components of the alveolar basement membrane to cells of the immune system, initiating an immune response [21]. The induction of vasculitis seems multifactorial, with interplay of environmental factors and genetic predisposition creating the environment for development of disease [22].

#### **4.2. Kidney-lung crosstalk**

**3.** pauci-immune ( only circulating ANCA) – type III crescentic GN

The underlying pulmonary lesions (necrotic pulmonary capillaritis) are clinically expressed as diffuse alveolar haemorrhage. Disruption and degradation of the pulmonary capillary wall and interstitial matrix results in vessel wall destruction and necrosis. Cordier and Cottin [18] found capillaritis as the most common pathological finding (60%) in ANCA-related vasculitides with pulmonary complications. However, capillaritis was not observed in all patients. It is likely that vessel inflammations may be overlooked in some cases if not

According to recommendations of Jennette and Falk [20] an accurate precise clinical diagnosis usually requires the integration of many different types of data, including clinical signs and symptoms, associated diseases, histological pattern of inflammation (eg, granulomatous versus necrotizing), immunopathological features (e.g. presence and composition of vascular immunoglobulin deposits), and serological findings (cryoglobulins, hypocomplementemia, hepatitis B antibodies, hepatitis C antibodies, ANCAs, anti–GBM antibodies, ANA). Specific diagnosis of a vasculitis is very important because the prognosis and appropriate therapy vary substantially among different types of vasculitis. Many attempts to re-evaluate and to refine the present nomenclature of vasculitis were done, however the complexity of vasculitic syndromes, as well as of pulmonary-renal syndrome complicates not only the estimation of

appropriate diagnosis, differential diagnosis but also hampers effective treatment.

Potentially accepted immunopathological mechanisms of pulmonary-renal syndrome involve antiglomerular basement membrane antibodies, antineutrophil cytoplasm antibodies, generation of immunocomplexes, activation of complement and haemocoagulation. Due to different pathogenesis of pulmonary-renal syndrome in various clinical diagnoses, common immunopathological features (except systemic inflammation) could not be specified. As seen in most inflammatory diseases, the systemic response to insult may be as important as the

The current understanding of autoimmune disorders suggests that some environmental factors initiate disease in a genetically susceptible individual. The importance of genetic factors, especially the genes of major histocompatibility complex has been increasingly recognized in determining susceptibility to autoimmune diseases. Because of the low incidence of all nosological entities of pulmonary-renal syndrome, it is not possible to examine the

In pulmonary-renal syndrome, as in most autoimmune disorders, the precise initiating events are not known. Exposure to hydrocarbons is known to cause damage to pulmonary/renal

inheritance of genes within affected families predisposing to this disease.

**3.2. Pulmonary pathology**

76 Updates in the Diagnosis and Treatment of Vasculitis

specifically searched [19].

**4. Immunopathogenesis**

**4.1. Genetical and environmental influence**

initial stimulus.

Newexperimentaldatahaveemergedinrecentyearsfocusingontheinteractiveeffectsofkidney and lung dysfunction, and these studies have highlighted the pathophysiological importance of proinflammatory and other immunologic pathways as well as the complex nature of interor‐ gancrosstalk.Becausepulmonaryandrenaldysfunctionfrequentlycoexist,the effectsoffailure of either organ are particularly relevant to the function of the other. Evidence suggests that deleteriouskidney-lunginteractionsor crosstalkrise,atleastinpart,duetothelossofthenormal balanceofimmune,inflammatoryandsolublemediatormetabolism.Theseprocessesoccurafter severe insults and cause induction of organ injury [23]. Organ crosstalk is a consequence of both directlossofnormalfunctionandinflammatorydysregulationresultingfrombothorganfailure. Cellular(e.g.neutrophils) aswell as solublemediators (cytokines) contribute tothe inflammato‐ ry dysregulation under these circumstances [24].

#### **4.3. Immunology of inflammation**

Interactions between inflammatory cells and damaged endothelium end in vessel inflammation (vasculitis), the major manifestation of all clinical entities. Cytokines, chemokines (such as IL-8) complement components, circulating immune complexes, and antibodies can be primary determinants of initiators of endothelial cell damage. When focusing on two basic clinical representatives – Goodpasture disease and granulomatosis with polyangiitis, damage of the vessel wall of glomeruli and alveolar capillaries are caused by antigen-stimulated white blood cells, anti-GBM antibodies and ANCA, respectively. In anti-GBM and ANCA vasculitis, the pathologic finding of focal, lytic necrotizing injury suggests highly effective local activation of neutrophils and monocytes with release of oxidants and proteases that are neutralized beyond the site of injury [13].

Concurrent ANCA and anti-GBM antibody production can be seen in selected patients, but reasonable explanation is unknown. It is possible that ANCA-related proteases damage or expose the nephritogenic epitopes in cr3 (IV) collagen in GBM, and this in turn leads to anti-GBM antibody production. It is unlikely that the crossreactivity between p-ANCA and anti-GBM antibodies is derived from the same autoantibody repertoire, because there does not appear to be a structural relationship between c-ANCA and a3 (IV) NC I collagen [25]. It is currently unclear whether there is any structural cross-reactivity between c-ANCA and anti-GBM antibodies [26].

#### **4.4. Cellular and humoral mechanisms**

Immunological mechanisms involved in initiation and prolongation of inflammatory state could be divided to two groups: cellular mechanisms (activity of immune-competent cells) and humoral mechanisms (proteins, mediators). Inflammation is tightly connected also to oxidative stress either through increased formation of ROS and/or the decreased activity of antioxidant systems.

"endothelialitis". Inhibition of NFkB activity by a decoy-oligonucleotide prevented

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79

**4.** The complement system probably plays an essential role in the initiation and propagation phases of vasculitis. Specifically the pneumococcal C-polysaccharide-reactive protein (CRP), synthesized after trauma and infection, can potently activate the complement cascade leading to activation of endothelial cells with increased expression of adhesion

Above mentioned pathogenetic mechanisms of vasculitides seem to be important and com‐ mon factors for the generation and maintenance of vascular inflammation; nevertheless these factorsareonlypartofthespectrumofdifferenthumoralandcellularresponses invasculitis[29].

Acute onset of severe pulmonary-renal syndrome is closely connected with ischemiareperfusion injury when both innate and adaptive immunity contribute to their pathogenesis. Kidney resident cells promote inflammation after ischemic/reperfusion injury by increasing endothelial cell adhesion molecule expression and vascular permeability. Kidney epithelial cells bind complement and express toll-like receptors and resident and infiltrating cells produce cytokines/chemokines. Early activation of kidney dendritic cells initiates a cascade of events leading to accumulation of interferon-γ-producing neutrophils, infiltrating macrophages, CD4+ T cells, B cells and invariant natural killer T cells. Bajwa et al. [30] recently implicated the IL23/IL17 pathway in kidney ischemic/reperfusion injury, as well as the importance that T-regulatory cells can directly suppress the early innate inflammation, induced by ischemia/reperfusion, in an IL-10 dependent manner. Following the initial early phase of inflammation, the late phase involves infiltration of anti-inflammatory cells including regulatory T cells, alternatively activated macrophages and stem cells leading to attenuation

Another possible connection between kidney and lung inflammation as a part of the systemic inflammatory pathway describe Campanholle et al. [31]. They concluded that pro-inflammato‐ rymediators,TNF-alfa,IL-1βandMCP-1,releasedbytheischemickidneymightreachthelungs, induce inflammation, up-regulate COX-2 and iNOS expressions, and ultimately contribute to

TNF-alpha, a potent pro-inflammatory cytokine belongs to the group of mediators that activate leukocytes and endothelial cells. Neutrophils, other leukocytes, and platelets adhere via cognate receptors to the pulmonary endothelium. Activated neutrophils release proteases, leukotrienes, reactive oxygen intermediates, and other inflammatory molecules that amplify the inflammatory response. ROS and proteases can directly damage alveolar–capillary membrane integrity [32]. On the other side the lectin-like domain of TNF-alpha has positive effects on permeability of the epithelial-endothelial barrier in the lungs. This domain is able

the accumulation and to activation of neutrophils and mononuclear cells.

activation of endothelial cells in reperfusion injury and vascular rejection.

molecules.

**4.5. Ischemic/reperfusion injury**

of inflammation and initiation of repair.

**4.6. TNFalfa – proinflammatory cytokine**

It should be postulated in the view of common association in all disease entities that the damage is caused by free radical formation of injured tissues. Local release of inflammatory cytokines and chemokines further activate endothelial cells to upregulate soluble adhesion molecules, enhanced activation of neutrophils and generation of reactive oxygen species which serve to amplify the initial inflammation leading to dysregulated apoptosis, secondary necrosis and overt vascular injury.

The immune system may target the tissues due to structural alterations in proteins or cell surfaces. Finally, the production of necessary anti-inflammatory factors may be impaired after hypoxia. Initiating signals – triggers of inflammation can activate the inflammatory process by several mechanisms that may occur simultaneously [27]:


Binding of ANCA to neutrophil membranes activates the cells leading to the release of lytic enzymes, chemoatractant interleukin-8 and oxygen free radicals. Neutrophils subsequently aggregate on endothelium causing inflammation and damage to the vasculature. Still, it remains unclear in many diseases whether or not ANCAs are simply playing a bystander role in the inflammatory cascade or directly driving vasculitic inflammation [28].

Gröne [29] describes except of the role of ANCAs, other immunopathogenetic factors important in vasculitides. They include innate immunity factors, transcription factors such as NFkB, endothelial cytoprotective agents such as NO. In summary:


"endothelialitis". Inhibition of NFkB activity by a decoy-oligonucleotide prevented activation of endothelial cells in reperfusion injury and vascular rejection.

**4.** The complement system probably plays an essential role in the initiation and propagation phases of vasculitis. Specifically the pneumococcal C-polysaccharide-reactive protein (CRP), synthesized after trauma and infection, can potently activate the complement cascade leading to activation of endothelial cells with increased expression of adhesion molecules.

Above mentioned pathogenetic mechanisms of vasculitides seem to be important and com‐ mon factors for the generation and maintenance of vascular inflammation; nevertheless these factorsareonlypartofthespectrumofdifferenthumoralandcellularresponses invasculitis[29].

#### **4.5. Ischemic/reperfusion injury**

humoral mechanisms (proteins, mediators). Inflammation is tightly connected also to oxidative stress either through increased formation of ROS and/or the decreased activity of

It should be postulated in the view of common association in all disease entities that the damage is caused by free radical formation of injured tissues. Local release of inflammatory cytokines and chemokines further activate endothelial cells to upregulate soluble adhesion molecules, enhanced activation of neutrophils and generation of reactive oxygen species which serve to amplify the initial inflammation leading to dysregulated apoptosis, secondary necrosis and

The immune system may target the tissues due to structural alterations in proteins or cell surfaces. Finally, the production of necessary anti-inflammatory factors may be impaired after hypoxia. Initiating signals – triggers of inflammation can activate the inflammatory process

**1.** Passively released factors from injured or exposed cells due to breakdown of cellular

**2.** Stress or injury (hypoxia) can induce the active synthesis of pro-inflammatory signals. **3.** Immune system receptors (cellular, humoral) may recognize altered or exposed surface

**4.** Damaged cells have decreased expression of inhibitory (anti-inflammatory) factors

Binding of ANCA to neutrophil membranes activates the cells leading to the release of lytic enzymes, chemoatractant interleukin-8 and oxygen free radicals. Neutrophils subsequently aggregate on endothelium causing inflammation and damage to the vasculature. Still, it remains unclear in many diseases whether or not ANCAs are simply playing a bystander role

Gröne [29] describes except of the role of ANCAs, other immunopathogenetic factors important in vasculitides. They include innate immunity factors, transcription factors such as

**1.** ANCA may be directed against several antigens, in the majority of cases against proteinase-3 and myeloperoxidase. The complex of proteinase-3 and ANCA leads to an increased expression of CD14, CD18 and an elevated synthesis of cytokines and chemokines such as interleukin 1, interleukin 8 in monocytes. In addition granulocytes generate reactive oxygen species, ANCA may also bind to a surface glycoprotein (gp130) expressed on glomerular and peritubular endothelia in the kidney. Thus the activation of granulocytes, monocytes and endothelial cells by ANCA may be a critical step in the

**3.** The transcription factor complex NFkB is a key regulatory transcription factor for the expression of genes and proteins associated with acute inflammatory processes and

permitting uncontrolled activation of inflammatory cells or systems.

in the inflammatory cascade or directly driving vasculitic inflammation [28].

NFkB, endothelial cytoprotective agents such as NO. In summary:

initiation phases of vasculitis, ultimately leading to apoptosis. **2.** NO is cytoprotective for endothelial cells in low concentrations.

by several mechanisms that may occur simultaneously [27]:

antioxidant systems.

78 Updates in the Diagnosis and Treatment of Vasculitis

overt vascular injury.

barriers.

structures.

Acute onset of severe pulmonary-renal syndrome is closely connected with ischemiareperfusion injury when both innate and adaptive immunity contribute to their pathogenesis. Kidney resident cells promote inflammation after ischemic/reperfusion injury by increasing endothelial cell adhesion molecule expression and vascular permeability. Kidney epithelial cells bind complement and express toll-like receptors and resident and infiltrating cells produce cytokines/chemokines. Early activation of kidney dendritic cells initiates a cascade of events leading to accumulation of interferon-γ-producing neutrophils, infiltrating macrophages, CD4+ T cells, B cells and invariant natural killer T cells. Bajwa et al. [30] recently implicated the IL23/IL17 pathway in kidney ischemic/reperfusion injury, as well as the importance that T-regulatory cells can directly suppress the early innate inflammation, induced by ischemia/reperfusion, in an IL-10 dependent manner. Following the initial early phase of inflammation, the late phase involves infiltration of anti-inflammatory cells including regulatory T cells, alternatively activated macrophages and stem cells leading to attenuation of inflammation and initiation of repair.

#### **4.6. TNFalfa – proinflammatory cytokine**

Another possible connection between kidney and lung inflammation as a part of the systemic inflammatory pathway describe Campanholle et al. [31]. They concluded that pro-inflammato‐ rymediators,TNF-alfa,IL-1βandMCP-1,releasedbytheischemickidneymightreachthelungs, induce inflammation, up-regulate COX-2 and iNOS expressions, and ultimately contribute to the accumulation and to activation of neutrophils and mononuclear cells.

TNF-alpha, a potent pro-inflammatory cytokine belongs to the group of mediators that activate leukocytes and endothelial cells. Neutrophils, other leukocytes, and platelets adhere via cognate receptors to the pulmonary endothelium. Activated neutrophils release proteases, leukotrienes, reactive oxygen intermediates, and other inflammatory molecules that amplify the inflammatory response. ROS and proteases can directly damage alveolar–capillary membrane integrity [32]. On the other side the lectin-like domain of TNF-alpha has positive effects on permeability of the epithelial-endothelial barrier in the lungs. This domain is able to blunt ROS production in pulmonary artery endothelial cells under hypoxia and reoxygenation, and reduce ROS content in inflammatory conditions [33].

During early stage of human septic shock Spapen et al. [43] founded massive decrease of IL-8 after N-acetylcystein (NAC) administration. According to that finding, Park et al. [44] supposes

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In Goodpasture´s syndrome normal exposure of epitopes by self-limited generation of ROS is not itself sufficient to launch a fatal autoimmune response. ROS can be produced in response to various normal stimuli such as mediators of inflammation, environmental toxins, de-novo respiratory bursts. Excessive ROS may influence GBM degradation by proteolytic enzymes [45]. Therefore, the presence of ROS in the microenvironment around the GBM can likely activate several pathways of protein modification in renal, as well as in pulmonary tissue in Goodpasture´s syndrome. Kalluri et al. [46] suggest that ROS can alter the hexameric structure of type IV collagen to expose or destroy selectively immunologic epitopes embedded in basement membrane. The reasons for autoimmunity in Goodpasture syndrome may lie in an age-dependent deterioration in inhibitor function modulating oxidative damage to structural molecules. ROS therefore may play an important role in shaping post-translational epitope

Natural antibodies produced by B-cells may play a role in prevention of pathological autoimmune reactions by binding to microbial epitopes that are similar or identical to self-

Bacterial superantigens trigger the activation of autoreactive cells such as toxic shock syndrome toxin 1 and Staphylococcal enterotoxins. These superantigens are generally

Under certain conditions, T helper type cells can differentiate into regulatory T cells producing immunosuppressive cytokines such as transforming growth factor-ß and IL-10. These regulatory T cells representing about 5% to 10% of CD4+ T cells in the steady state, play a central role in immune homeostasis and in preventing autoimmune diseases in general [50, 51]. Regulatory T cells exist naturally and are called natural regulatory T cells expressing CD25 and Foxp3. T cells can also convert into regulatory T cells upon certain antigen recognition and are called antigen-specific regulatory T cells that secrete IL-10 and/or transforming growth factor-β. Indeed, regulatory T cells are required to control infection-induced immunity in a

Recently discovered regulatory Th17-cells and cell-derived cytokines play an important role in the pathogenesis of several autoimmune/inflammatory diseases including vasculitides. In Wegener´s granulomatosis, regulatory T-cells display impaired suppressor activity potentially favouring inflammation and break of tolerance [53]. Th17-cells produce several cytokines such as IL-17, IL-21, IL-22, CCL-20 which induce massive inflammatory tissue reactions and these cytokines also stimulate nonimmune cells (fibroblasts, endothelial and epithelial cells) to the production of proinflammatory mediators (IL-6, TNF-alfa, prostaglandins, NO, MMP and

considered to be the triggers of exacerbation in Wegener´s granulomatosis [48, 49].

another possible mechanism of NAC effect - through inhibition of IL-8.

diversity or neoantigen formation in organ tissues.

host, including autoimmunity inhibition [52].

**4.8. Natural antibodies**

antigens [47].

**4.9. Role of Th 17**

TNF-alpha - an important cytokine involved in pathogenesis of inflammation, is an example of a "moonlighting protein", with differential activities mediated by its receptor-binding versus its lectin-like domains, which opens the possibility to design and develop more sophisticated therapeutic regimens for patients with increased permeability of the epithelialendothelial barrier of the lung, which in pulmonary-renal syndrome can occur. However, in the future, more research is needed in order to reveal the underlying mechanisms of TNF's protective versus deleterious effects [34].

#### **4.7. Reactive oxygen species and nitric oxide engagement**

Among enzymes incorporated in the free radical formation, eNOS (NOS3) has been shown to inhibit vascular inflammation in many different model systems, but its role in the pathogenesis of vasculitis has not been elucidated yet. According to Schoeb et al. [35] eNOS serves as a negative regulator of vasculitis in experimental (mice) model of pulmonary-renal syndrome and they further suggest that nitric oxide produced by this enzyme may be critical for inhibiting lesion formation and vascular damage in human vasculitides. Derangements in the key oxidative stress enzymes, nitric oxide synthase and heme oxygenase may also facilitate distant organ dysfunction. Disordered NO metabolism in the setting of inflammation is well established. While the cause of this dysregulation is not entirely clear, asymmetric dimethyarginine seems to play a significant role [36, 37]. Asymmetric dimethyarginine is an inhibitor of endothelial NO synthase and shifts NO metabolism toward production of oxygenbased free radicals [38]. MPO released from activated neutrophils are involved in the formation of NO-derived reactive oxygen species [39]. ROS produced by macrophages in combination with reactive nitrogen intermediates cause protein nitration in endothelial cells [40]. This could be related to activation of cytokines produced by macrophages to elicit proinflammatory and prothrombotic responses in endothelial cells [41]. MPO produces a highly deleterious reactive oxygen species, the hypochlorous acid (HOCl). Anti-MPO antibodies from patients with small vessel vasculitis (MPA) can trigger the release of MPO by neutrophils and monocytes. Anti-MPO antibodies can activate MPO to generate an oxidative stress deleterious for the endothelium.

Guilpain et al. [42] recently demonstrated that MPA sera with anti-MPO antibodies activated MPO in vitro, and generated free radicals (hypochlorous acid), whereas sera from MPA patients with no anti-MPO antibodies or healthy individuals did not. Free oxygen radical production and endothelial lysis were abrogated by N-acetylcysteine (NAC), an antioxidant molecule through the augmentation of glutathione biosynthesis. N-acetylcysteine significantly reduced the activation of myeloperoxidase and improved the survival of endothelial cells exposed to byproducts of myeloperoxidase activation. Thus, anti-MPO antibodies could play a pathogenic role in vivo by triggering an oxidative burst leading to severe endothelial damages.

During early stage of human septic shock Spapen et al. [43] founded massive decrease of IL-8 after N-acetylcystein (NAC) administration. According to that finding, Park et al. [44] supposes another possible mechanism of NAC effect - through inhibition of IL-8.

In Goodpasture´s syndrome normal exposure of epitopes by self-limited generation of ROS is not itself sufficient to launch a fatal autoimmune response. ROS can be produced in response to various normal stimuli such as mediators of inflammation, environmental toxins, de-novo respiratory bursts. Excessive ROS may influence GBM degradation by proteolytic enzymes [45]. Therefore, the presence of ROS in the microenvironment around the GBM can likely activate several pathways of protein modification in renal, as well as in pulmonary tissue in Goodpasture´s syndrome. Kalluri et al. [46] suggest that ROS can alter the hexameric structure of type IV collagen to expose or destroy selectively immunologic epitopes embedded in basement membrane. The reasons for autoimmunity in Goodpasture syndrome may lie in an age-dependent deterioration in inhibitor function modulating oxidative damage to structural molecules. ROS therefore may play an important role in shaping post-translational epitope diversity or neoantigen formation in organ tissues.

#### **4.8. Natural antibodies**

to blunt ROS production in pulmonary artery endothelial cells under hypoxia and

TNF-alpha - an important cytokine involved in pathogenesis of inflammation, is an example of a "moonlighting protein", with differential activities mediated by its receptor-binding versus its lectin-like domains, which opens the possibility to design and develop more sophisticated therapeutic regimens for patients with increased permeability of the epithelialendothelial barrier of the lung, which in pulmonary-renal syndrome can occur. However, in the future, more research is needed in order to reveal the underlying mechanisms of TNF's

Among enzymes incorporated in the free radical formation, eNOS (NOS3) has been shown to inhibit vascular inflammation in many different model systems, but its role in the pathogenesis of vasculitis has not been elucidated yet. According to Schoeb et al. [35] eNOS serves as a negative regulator of vasculitis in experimental (mice) model of pulmonary-renal syndrome and they further suggest that nitric oxide produced by this enzyme may be critical for inhibiting lesion formation and vascular damage in human vasculitides. Derangements in the key oxidative stress enzymes, nitric oxide synthase and heme oxygenase may also facilitate distant organ dysfunction. Disordered NO metabolism in the setting of inflammation is well established. While the cause of this dysregulation is not entirely clear, asymmetric dimethyarginine seems to play a significant role [36, 37]. Asymmetric dimethyarginine is an inhibitor of endothelial NO synthase and shifts NO metabolism toward production of oxygenbased free radicals [38]. MPO released from activated neutrophils are involved in the formation of NO-derived reactive oxygen species [39]. ROS produced by macrophages in combination with reactive nitrogen intermediates cause protein nitration in endothelial cells [40]. This could be related to activation of cytokines produced by macrophages to elicit proinflammatory and prothrombotic responses in endothelial cells [41]. MPO produces a highly deleterious reactive oxygen species, the hypochlorous acid (HOCl). Anti-MPO antibodies from patients with small vessel vasculitis (MPA) can trigger the release of MPO by neutrophils and monocytes. Anti-MPO antibodies can activate MPO to generate an oxidative stress deleterious for the

Guilpain et al. [42] recently demonstrated that MPA sera with anti-MPO antibodies activated MPO in vitro, and generated free radicals (hypochlorous acid), whereas sera from MPA patients with no anti-MPO antibodies or healthy individuals did not. Free oxygen radical production and endothelial lysis were abrogated by N-acetylcysteine (NAC), an antioxidant molecule through the augmentation of glutathione biosynthesis. N-acetylcysteine significantly reduced the activation of myeloperoxidase and improved the survival of endothelial cells exposed to byproducts of myeloperoxidase activation. Thus, anti-MPO antibodies could play a pathogenic role in vivo by triggering an oxidative burst leading to severe endothelial

reoxygenation, and reduce ROS content in inflammatory conditions [33].

protective versus deleterious effects [34].

80 Updates in the Diagnosis and Treatment of Vasculitis

endothelium.

damages.

**4.7. Reactive oxygen species and nitric oxide engagement**

Natural antibodies produced by B-cells may play a role in prevention of pathological autoimmune reactions by binding to microbial epitopes that are similar or identical to selfantigens [47].

Bacterial superantigens trigger the activation of autoreactive cells such as toxic shock syndrome toxin 1 and Staphylococcal enterotoxins. These superantigens are generally considered to be the triggers of exacerbation in Wegener´s granulomatosis [48, 49].

#### **4.9. Role of Th 17**

Under certain conditions, T helper type cells can differentiate into regulatory T cells producing immunosuppressive cytokines such as transforming growth factor-ß and IL-10. These regulatory T cells representing about 5% to 10% of CD4+ T cells in the steady state, play a central role in immune homeostasis and in preventing autoimmune diseases in general [50, 51]. Regulatory T cells exist naturally and are called natural regulatory T cells expressing CD25 and Foxp3. T cells can also convert into regulatory T cells upon certain antigen recognition and are called antigen-specific regulatory T cells that secrete IL-10 and/or transforming growth factor-β. Indeed, regulatory T cells are required to control infection-induced immunity in a host, including autoimmunity inhibition [52].

Recently discovered regulatory Th17-cells and cell-derived cytokines play an important role in the pathogenesis of several autoimmune/inflammatory diseases including vasculitides. In Wegener´s granulomatosis, regulatory T-cells display impaired suppressor activity potentially favouring inflammation and break of tolerance [53]. Th17-cells produce several cytokines such as IL-17, IL-21, IL-22, CCL-20 which induce massive inflammatory tissue reactions and these cytokines also stimulate nonimmune cells (fibroblasts, endothelial and epithelial cells) to the production of proinflammatory mediators (IL-6, TNF-alfa, prostaglandins, NO, MMP and chemokines [54]. New results, showing the possibility that regulatory Th17 cells and corresponding cytokines (IL-17, IL-23) involved in the pathogenesis of GPS as well in WG might be used for the directed therapy of pulmonary-renal syndrome in the future.

conceivably delay recovery. Strategies that prevent the initiation of inflammation by targeting the earliest signals or recognition of the injured tissue may be of particular therapeutic benefit in

Immunological Mechanisms and Clinical Aspects in Pulmonary-Renal Syndrome: A Review

http://dx.doi.org/10.5772/55181

83

The etiology of pulmonary-renal syndrome could be associated with variety of diseases. A possible classification based on clinical symptomatology and histopathology is described in

**Etiology of pulmonary-renal syndrome**

Wegener's granulomatosis Churg-Strauss syndrome Cryoglobulinemia Henoch-Schönlein purpura Behçet's syndrome Microscopic polyarteritis

Polymyositis or dermatomyositis Progressive systemic sclerosis

**5. Semi-systemic (pathologic/clinical) classification**

RA SLE MCTD

"Catastrophic" APS

IgA nephropathy

Infection Coagulopathy Heart failure

Goodpasture´s disease

Post-renal transplation failure Idiopathic pulmonary-renal syndrome

It is very important to establish an early diagnosis based on clinical vigilance, contemporary diagnostic laboratory support (immunology and biopsy) for the pulmonary-renal syndrome

Idiopathic immune complex glomerulonephritis

Rapidly progressive glomerulonephritis with heart failure

Drugs (D-penicillamine, propylthiouracil, carbimazole, cocaine, …)

these conditions [25].

**Cause Disease**

**Table 2.** Etiology of pulmonary-renal syndrome

**6. Clinical involvement**

Table 2.

Systemic vasculitides

Connective tissue disorders

Renal disorders

Other

Vasculitis (especially Wegener´s granulomatosis) is associated with bacterial infection, in particular nasal occurence of Staphylococcus aureus. Infection may play a role in the induction of autoimmunity as well as in the effector phase of the disease. In this relation Tadema et al. [55] emphasize the role of innate immunity that is involved in the development of a Th17 driven immune response, consistent with skewing towards a Th17 T cell phenotype that has been observed in Wegener's granulomatosis. Their findings shed new light on the potential role of γ/δ T cells in host defense and inflammatory diseases, provide important new information on the pathogenic role of IL-23 and IL-1β, and underline the importance of targeting these cytokines in the development of new therapeutic interventions against many autoimmune diseases.

Sutton et al. [56] demonstrated that γ/δ T cells activated by IL-1β and IL-23 are an important source of innate IL-17 and IL-21 and provide an alternative mechanism whereby IL-1 and IL-23 may mediate autoimmune inflammation.

In other study Ooi et al. [57] suggested the importance of IL-23, a key cytokine in the induction andmaintenanceofautoimmuneresponses,inTh1responsesthatcouldplayaroleinsomeforms of glomerulonephritis especially in anti-GBM (Goodpasture) disease. This experimental work emphasizes potential mechanisms in the treatment of several forms of glomerulonephritis.

Accumulating data from animal models support a role for Th17 cells and their cytokines in various autoimmune and inflammatory processes. Emerging data from running clinical trials indicate the importance of Th17 cells in such immunological processes, too. Future studies will allow us to evaluate the role of each cytokine independently in contributing to human diseases with immune-mediated pathologies and to design optimal cytokine-targeted therapies for these diseases [58].

#### **4.10. Perspectives of treatment – modulation of inflammation**

After careful analysis of numerous animal studies demonstrating the importance of inflamma‐ tion in the pathogenesis of pulmonary-renal syndrome, as well as the clinical correlates demon‐ stratingactivationofthe same systems inpatientswithsystemic autoimmunity,there is a reason to hope that different modalities of anti-inflammatory treatment could ameliorate the course of the disease. The inflammatory process is however extremely complex due to its multifactorial etiologyandconsideringalsointhecontextofdifferencesamongsystems [59].Forexamplerenal failure involves complex host-kidney interactions in which the inflammatory state of the host contributes to the development of renal failure and injury of the inflamed tissue further modu‐ lates the inflammatory state of the host. One of the greatest obstacles to effective treatment is establishing the diagnosis as early as possible based on all available diagnostic procedures, including invasive ones. Earlier and more reliable identification of clinical signs and laboratory markershasbecomeanimportanttoolinrelationtoestablisheffectivetreatmentmodalities.Once the inflammatory response has been set in motion, treatment may be ineffective and could conceivably delay recovery. Strategies that prevent the initiation of inflammation by targeting the earliest signals or recognition of the injured tissue may be of particular therapeutic benefit in these conditions [25].
